Cd70 combination therapy

ABSTRACT

The present invention relates to combination therapies for the treatment of malignancy, particularly myeloid malignancy such as acute myeloid leukemia (AML). The combination therapies may include an antibody molecule that binds to CD70 and at least one antibody molecule that binds to a leukemic stem cell target. Preferred leukemic stem cell targets are TIM-3, IL1R3/IL1RAP and CD47.

RELATED APPLICATIONS

This application claims benefit of Great Britain Patent Application No.1800649.4, filed Jan. 16, 2018, the entire contents of which areincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 16, 2019, isnamed 608245_AGX5-038_Sequence_Listing.txt and is 59,685 bytes in size.

FIELD OF THE INVENTION

The present invention relates to combination therapies for the treatmentof malignancy, particularly myeloid malignancy such as acute myeloidleukemia (AML). The combination therapies may include an antibodymolecule that binds to CD70 and at least one antibody molecule thatbinds to a leukemic stem cell (LSC) target. Preferred leukemic stem celltargets are TIM-3, IL1R3/IL1RAP and CD47. Alternatively or in addition,the combination therapies may include an antibody molecule that binds toCD70 and an agent that inhibits SIRPα signaling. The combinationtherapies may further include an additional anti-cancer agent, forexample an agent used for the treatment of AML such as azacitidine ordecitabine.

BACKGROUND TO THE INVENTION

In recent years, the development of new cancer treatments has focused onmolecular targets, particularly proteins, implicated in cancerprogression. The list of molecular targets involved in tumor growth,invasion and metastasis continues to expand, and includes proteinsoverexpressed by tumor cells as well as targets associated with systemssupporting tumor growth such as the vasculature and immune system. Thenumber of therapeutic or anti-cancer agents designed to interact withthese molecular targets also continues to increase, and a large numberof targeted cancer medicines are now approved for clinical use with manymore in the developmental pipeline.

Immunotherapy is a particularly interesting approach to cancertreatment. This form of therapy seeks to harness the power of the body'sown immune system to control tumor growth. The immune system is highlycomplex including a multitude of different cell types and in healthyindividuals, these different cell populations are subject to tightcontrol. During cancer development, tumors typically evolve ways toevade detection and elimination by the host immune system, for exampleby downregulation of natural killer (NK) cell activators, reducedexpression of MHC class I proteins by the tumor cells, T cell anergyand/or the upregulation of immunosuppressive regulatory T cells (Tregs).Immunotherapy aims to reverse the immunosuppressive tumor environmentthereby aiding the body in mounting an effective anti-tumor response.

CD70 has been identified as a molecular target of particular interestowing to its constitutive expression on many types of hematologicalmalignancies and solid carcinomas (Junker et al. (2005) J Urol.173:2150-3; Sloan et al. (2004) Am J Pathol. 164:315-23; Held-Feindt andMentlein (2002) Int J Cancer 98:352-6; Hishima et al. (2000) Am J SurgPathol. 24:742-6; Lens et al. (1999) Br J Haematol. 106:491-503;Boursalian et al. (2009) Adv Exp Med Biol. 647:108-119; Wajant H. (2016)Expert Opin Ther Targets 20(8):959-973). CD70 is a type II transmembraneglycoprotein belonging to the tumor necrosis factor (TNF) superfamily,which mediates its effects through binding to its cognate cell surfacereceptor, CD27. Both CD70 and CD27 are expressed by multiple cell typesof the immune system, and the CD70-CD27 signaling pathway has beenimplicated in the regulation of several different aspects of the immuneresponse. This is reflected in the fact that CD70 overexpression occursin various auto-immune diseases including rheumatoid and psoriaticarthritis and lupus (Boursalian et al. (2009) Adv Exp Med Biol.647:108-119; Han et al. (2005) Lupus 14(8):598-606; Lee et al. (2007) JImmunol. 179(4):2609-2615; Oelke et al. (2004) Arthritis Rheum.50(6):1850-1860).

CD70 expression has been linked to poor prognosis for several cancersincluding B cell lymphoma, renal cell carcinoma and breast cancer(Bertrand et al. (2013) Genes Chromosomes Cancer 52(8):764-774;Jilaveanu et al. (2012) Hum Pathol. 43(9):1394-1399; Petrau et al.(2014) J Cancer 5(9):761-764). CD70 expression has also been found onmetastatic tissue in a high percentage of cases indicating a key rolefor this molecule in cancer progression (Jacobs et al. (2015) Oncotarget6(15):13462-13475). Constitutive expression of CD70 and its receptorCD27 on tumor cells of hematopoietic lineage has been linked to a roleof the CD70-CD27 signaling axis in directly regulating tumor cellproliferation and survival (Goto et al. (2012) Leuk Lymphoma53(8):1494-1500; Lens et al. (1999) Br J Haematol. 106(2); 491-503;Nilsson et al. (2005) Exp Hematol. 33(12):1500-1507; van Doorn et al(2004) Cancer Res. 64(16):5578-5586).

Upregulated CD70 expression on tumors, particularly solid tumors that donot co-express CD27, also contributes to immunosuppression in the tumormicroenvironment in a variety of ways. For example, CD70 binding to CD27on regulatory T cells has been shown to augment the frequency of Tregs,reduce tumor-specific T cell responses and promote tumor growth in mice(Claus et al. (2012) Cancer Res. 72(14):3664-3676). CD70-CD27 signalingcan also dampen the immune response by tumor-induced apoptosis ofT-lymphocytes, as demonstrated in renal cell carcinoma, glioma andglioblastoma cells (Chahlavi et al. (2005) Cancer Res. 65(12):5428-5438;Diegmann et al. (2006) Neoplasia 8(11):933-938); Wischusen et al. (2002)Cancer Res 62(9):2592-2599). Finally, CD70 expression has also beenlinked to T cell exhaustion whereby the lymphocytes adopt a moredifferentiated phenotype and fail to kill the tumor cells (Wang et al.(2012) Cancer Res 72(23):6119-6129; Yang et al. (2014) Leukemia28(9):1872-1884).

Given the importance of CD70 in cancer development, CD70 is anattractive target for anti-cancer therapy, and antibodies targeting thiscell surface protein are in clinical development (Jacob et al. (2015)Pharmacol Ther. 155:1-10; Silence et al. (2014) mAbs 6(2):523-532).

SUMMARY OF INVENTION

The present invention is directed to combination therapies comprisingantibody molecules that bind to CD70. In the combination therapies ofthe invention, an antibody molecule that binds to CD70 is combined withat least one additional agent directed to a different target so as toachieve more effective cancer treatment. The agents with which the CD70antibody molecules may be combined include antibody molecules that bindto leukemic stem cell targets and/or agents that inhibit SIRPαsignaling.

It has been found that anti-CD70 antibodies are effective for thetreatment of myeloid malignancies, particularly acute myeloid leukemia(AML) and myelodysplastic syndrome (MDS). The present invention is basedon the use of antibody molecules that bind CD70 in combination withadditional agents that target malignant myeloid cells, particularlyleukemic stem cells.

In a first aspect, the present invention provides a combinationcomprising an antibody molecule that binds to CD70 and at least oneantibody molecule that binds to a leukemic stem cell target.

In certain embodiments, the leukemic stem cell target is selected fromthe group consisting of: TIM-3; Galectin-9; CD47; IL1RAP; LILRB2; CLL-1;CD123; CD33; SAIL; GPR56; CD44; E-selectin; CXCR4; CD25; CD32; PR1; WT1;ADGRE2; CCR1; TNFRSF1B and CD96. In preferred embodiments, the leukemicstem cell target is selected from TIM-3, Galectin-9, CD47, IL1RAP andLILRB2.

In certain embodiments, the antibody molecule that binds to CD70 isselected from: (i) an antibody molecule comprising a variable heavychain domain (VH) and a variable light chain domain (VL) comprising theheavy chain CDRs (HCDR3, HCDR2 and HCDR1) and light chain CDRs (LCDR3,LCDR2 and LCDR1): HCDR3 having an amino acid sequence comprising orconsisting of SEQ ID NO: 3; HCDR2 having an amino acid sequencecomprising or consisting of SEQ ID NO: 2; HCDR1 having an amino acidsequence comprising or consisting of SEQ ID NO: 1; LCDR3 having an aminoacid sequence comprising or consisting of SEQ ID NO: 7; LCDR2 having anamino acid sequence comprising or consisting of SEQ ID NO: 6; and LCDR1having an amino acid sequence comprising or consisting of SEQ ID NO: 5;(ii) an antibody molecule comprising a VH domain comprising an aminoacid sequence at least 70%, at least 80%, at least 90%, or at least 95%identical to SEQ ID NO:4 and a VL domain comprising an amino acidsequence at least 70%, at least 80%, at least 90%, or at least 95%identical to SEQ ID NO:8; or (iii) ARGX-110.

In certain embodiments, the combinations comprise, in addition to theantibody molecule that binds CD70, an antibody molecule that binds to afirst leukemic stem cell target and an antibody molecule that binds to asecond leukemic stem cell target, wherein the first and second leukemicstem cell targets are different. The first and/or second leukemic stemcell targets may be selected from the group consisting of: TIM-3;Galectin-9; CD47; IL1RAP; LILRB2; CLL-1; CD123; CD33; SAIL; GPR56; CD44;E-selectin; CXCR4; CD25; CD32; PR1; WT1; ADGRE2; CCR1; TNFRSF1B andCD96, preferably the group consisting of: TIM-3; Galectin-9; CD47;IL1RAP and LILRB2. In preferred embodiments, the first leukemic stemcell target is TIM-3 and the second leukemic stem cell target is CD47.In further preferred embodiments, the first leukemic stem cell target isTIM-3 and the second leukemic stem cell target is IL1 RAP.

For combinations of the invention comprising an antibody molecule thatbinds TIM-3 or an antibody molecule that binds IL1RAP, in certainembodiments, this antibody molecule results in reduced NF-κB signaling;reduced Wnt/β-catenin signaling; reduced stemness of AML cells; or acombination thereof. Alternatively or in addition, the antibody moleculethat binds TIM-3 may inhibit the interaction of TIM-3 with one or moreTIM-3 interacting proteins, optionally TIM-3 interacting proteinsselected from: CEACAM-1; HMGB-1; phosphatidylserine; Galectin-9; LILRB2;and combinations thereof.

In certain embodiments, the antibody molecule or antibody molecules thatbind to LSC target(s) is/are camelid-derived. For example, the antibodymolecules may be selected from one or more immune libraries obtained bya method comprising the step of immunizing a camelid, preferably allama, with the leukemic stem cell target(s). The antibody molecules maybe derived from camelid by immunizing an animal of a camelid specieswith the LSC target protein or a polypeptide fragment thereof or byimmunizing a camelid species with a mRNA or cDNA molecule expressing theLSC target protein or a polypeptide fragment thereof.

For combinations of the invention comprising an antibody molecule thatbinds CD47, in certain embodiments, the antibody molecule inhibits theinteraction between CD47 on the leukemic stem cells and SIRPα onphagocytic cells. The antibody molecule that binds CD47 mayalternatively or in addition increase phagocytosis of tumor cells.

In certain embodiments, the antibody molecules of the combination areindependently selected from the group consisting of: an IgG antibody; anantibody light chain variable domain (VL); an antibody heavy chainvariable domain (VH); a single chain antibody (scFv); a F(ab′)2fragment; a Fab fragment; an Fd fragment; an Fv fragment; a one-armed(monovalent) antibody; diabodies, triabodies, tetrabodies or anyantigen-binding molecule formed by combination, assembly or conjugationof such antigen binding fragments.

Regarding the formulation of the combination, in certain embodiments,the antibody molecules of the combination are combined in a singleantibody format, for example as a multispecific antibody, preferably abispecific antibody. Alternatively, the antibody molecules may beseparate but co-formulated in a single composition. For antibodymolecules co-formulated as a single composition, the ratio of thedifferent antibody molecules may be 1:1. Alternatively, the antibodymolecules may be present in different relative amounts. For example, forembodiments of the invention in which the combination comprises a firstantibody molecule that binds to CD70 and a second antibody molecule thatbinds to a LSC target, the ratio of first antibody molecule to secondantibody molecule may be 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, 5:1 etc. Inalternative embodiments, the antibody molecules are provided separately.

The antibody molecules of the combinations may possess one or moreeffector functions. In certain embodiments, at least one of the antibodymolecules: blocks its target's function completely or partially; and/orhas antibody-dependent cellular cytotoxicity (ADCC) activity; and/orcomprises a defucosylated antibody domain; and/or hascomplement-dependent cytotoxicity (CDC) activity; and/or hasantibody-dependent cellular phagocytosis (ADCP) activity.

The combinations of the invention may comprise one or more additionaltherapeutic agents, for example one or more additional anti-canceragents. In certain embodiments, the combination comprises an agent thatinhibits SIRPα signaling. The agent that inhibits SIRPα signaling may bean antibody molecule that binds to SIRPα and inhibits the interactionbetween CD47 and SIRPα or alternatively may be a SIRPα-antibody moleculefusion protein, for example a SIRPα-Fc fusion. In certain embodiments,the SIRPα-antibody molecule fusion protein comprises at least one of theantibody molecules of the combination. In particular embodiments, theSIRPα-antibody molecule fusion protein comprises the antibody moleculeof the combination that binds CD70.

In a further aspect, the present invention provides a combinationcomprising an antibody molecule that binds to CD70 and an agent thatinhibits SIRPα signaling.

The agent that inhibits SIRPα signaling may be selected from: (i) anantibody molecule that binds CD47 and inhibits the interaction betweenCD47 and SIRPα; (ii) an antibody molecule that binds SIRPα and inhibitsthe interaction between CD47 and SIRPα; (iii) a SIRPα-antibody moleculefusion protein, optionally a SIRPα-Fc fusion protein. In particularembodiments, the antibody molecule that binds to CD70 and the agent thatinhibits SIRPα signaling are combined into a single molecule, forexample a SIRPα-antibody molecule fusion protein wherein the antibodymolecule comprises or consists of the antibody molecule that binds CD70.In particular embodiments, the combinations of the second aspect of theinvention comprise at least one SIRPα V-like domain covalently linked tothe antibody molecule that binds to CD70. The linkage between the SIRPαV-like domain and the antibody molecule that binds to CD70 may bemediated via a linker.

In all aspects of the invention, in certain embodiments, the combinationcomprises at least one additional anti-cancer agent, for example atleast one agent for treating myeloid malignancy. In certain embodiments,the combinations herein comprise an additional agent for treating AML.In preferred embodiments, the combinations comprise a hypomethylatingagent, preferably azacitidine. Alternatively or in addition, thecombination may comprise a PD-1 inhibitor and/or a PD-L1 inhibitor.

In a further aspect, the present invention provides combinationsaccording to the first and second aspects of the invention for use inthe treatment of malignancy in a human subject. The present inventionalso provides a method for treating a malignancy in a human subject,said method comprising administering to the subject any of thecombinations according to the first or second aspects of the invention.

The present invention also provides an antibody molecule that binds toCD70 for use in the treatment of a malignancy in a human subject,wherein the antibody molecule is administered in combination with anantibody molecule that binds to a leukemic stem cell target. The presentinvention also provides an antibody molecule that binds to a leukemicstem cell target for use in the treatment of a malignancy in a humansubject, wherein the antibody molecule is administered in combinationwith an antibody molecule that binds to CD70.

The present invention also provides an antibody molecule that binds toCD70 for use in the treatment of malignancy in a human subject, whereinthe antibody molecule is administered in combination with an agent thatinhibits SIRPα signaling. The present invention also provides an agentthat inhibits SIRPα signaling for use in the treatment of malignancy ina human subject, wherein the agent is administered in combination withan antibody molecule that binds CD70.

Regarding the malignancies to be treated using combinations of theinvention, said malignancies may be (i) malignancies comprising theproduction of cancer progenitor or stem cells expressing CD70, CD27 orboth; (ii) malignancies comprising the production of cancer progenitoror stem cells expressing the LSC target to which at least one of theantibody molecules of the combination binds; (iii) myeloid malignancies;(iv) newly diagnosed myeloid malignancies; (v) relapsed or refractorymyeloid malignancies; (vi) myeloid malignancies selected from: acutemyeloid leukemia (AML), myelodysplastic syndromes (MDS),myeloproliferative neoplasms (MPN), chronic myeloid leukemia (CML), andchronic myelomonocytic leukemia (CMML). In a particularly preferredembodiment, the combinations of the invention are for the treatment ofacute myeloid leukemia (AML).

In certain embodiments, the methods further comprise monitoring of thepatient's blast count. The patient's peripheral blood and/or bone marrowcount may be reduced, for example reduced to less than 25%, for examplereduced to 5%, for example reduced to less than 5%, for example reducedto undetectable levels. In certain embodiments, the bone marrow blastcount is reduced to between 5% and 25% and the bone marrow blastpercentage is reduced by more than 50% as compared to pretreatment.

In certain embodiments, the methods induce at least a partial response.In certain embodiments, the methods induce a complete response,optionally with platelet recovery and/or neutrophil recovery. Themethods may induce transfusion independence of red blood cells orplatelets, or both, for 8 weeks or longer, 10 weeks or longer, or 12weeks or longer. In certain embodiments, the methods reduce themortality rate after a 30-day period or after a 60-day period.

In certain embodiments, the methods increase survival. For example, themethods may increase survival relative to the standard of care agent oragents used to treat the particular myeloid malignancy being treatedwith the combination. The methods may induce a minimal residual diseasestatus that is negative.

In certain embodiments, the methods further comprise a step ofsubjecting the subject to a bone marrow transplantation. Alternativelyor in addition, the methods may further comprise a step of administeringone or more additional anti-cancer agents. The one or more additionalanti-cancer agents may be selected from any agents suitable for thetreatment of myeloid malignancies, preferably AML. Preferred agents maybe selected from Venetoclax; Vyxeos; Idhifa (Enasidenib); and Rydapt(midostaurin).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the combined efficacy of an anti-CD70 antibody (ARGX-110)and anti-TIM-3 antibodies (1A11 and 2610) in mediatingantibody-dependent cellular phagocytosis (ADCP) against an AML cell line(BDCM).

FIG. 2 shows the combined efficacy of an anti-CD70 antibody (ARGX-110)and anti-IL1RAP antibodies (1F10, 1C1, 7E4G1E8 and 89412) in mediatingantibody-dependent cellular phagocytosis (ADCP) against AML cell lines(MV4-11, THP1 and U937).

FIGS. 3A and 3B show the combined efficacy of an anti-CD70 antibody(ARGX-110) and anti-CD47 antibodies (B6H12, CC2C6 and BRIC126) inmediating antibody-dependent cellular phagocytosis (ADCP) against AMLcell lines (MV4-11, THP1, GDM1 and U937).

FIG. 4 shows the combined efficacy of an anti-CD70 antibody (ARGX-110)and anti-TIM-3 antibodies (1A11 and 2B10) in mediatingcomplement-mediated cytotoxicity (CDC).

FIGS. 5A and 5B show the combined efficacy of an anti-CD70 antibody(ARGX-110) and anti-IL1RAP antibodies (1F10 and 1C1) in mediatingcomplement-mediated cytotoxicity (CDC). FIG. 5A shows CDC measured usingMV4-11 AML cells; FIG. 5B shows CDC measured using NOMO-1 AML cells.

FIGS. 6A and 6B show the combined efficacy of an anti-CD70 antibody(ARGX-110) and an anti-CD47 antibody (BRIC126) in mediatingcomplement-mediated cytotoxicity (CDC). FIG. 6A shows CDC measured usingMV4-11 AML cells; FIG. 6B shows CDC measured using NOMO-1 AML cells.

FIG. 7 shows the combined efficacy of an anti-CD70 antibody (ARGX-110)and an anti-TIM-3 antibody (2B10) in mediating antibody-dependentcellular cytotoxicity (ADCC) against an AML cell line (BDCM).

FIG. 8 shows the combined efficacy of an anti-CD70 antibody (ARGX-110)and an anti-IL1RAP antibody (1F10) in mediating complement-mediatedcytotoxicity (CDC) against an AML cell line (NOMO-1).

FIG. 9 shows the combined efficacy of an anti-CD70 antibody (ARGX-110)and an anti-CD47 antibody (CC2C6) in mediating complement-mediatedcytotoxicity (CDC) against an AML cell line (NOMO-1).

DETAILED DESCRIPTION

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one skilled in theart in the technical field of the invention.

“Combination therapy”—As used herein, the term “combination therapy”refers to a treatment in which a subject, for example a human subject,is given two or more therapeutic agents. The “combinations” describedherein are for use in combination therapy. The two or more therapeuticagents are typically administered so as to treat a single disease,herein a malignancy. In certain embodiments, the combination therapiesof the present invention utilize antibody molecules binding to differenttargets, specifically CD70 and a leukemic stem cell target, for exampleTIM-3, CD47 or IL1RAP. As described elsewhere herein, the antibodymolecules included in the combination therapies may be comprised withina single antibody (for example, a multispecific antibody), may beco-formulated or may be provided separately, for example as separatecompositions, for administration to a subject or patient in needthereof. In certain embodiments, the combination therapies of thepresent invention utilize an antibody molecule that binds to CD70 incombination with at least one agent that inhibits SIRPα signaling. Theantibody molecule that binds to CD70 may be combined with the agent thatinhibits SIRPα signaling into a single SIRPα-antibody molecule fusionprotein. Alternatively, the antibody molecule that binds to CD70 and theagent that inhibits SIRPαsignaling may be provided separately, forexample as separate compositions, for administration to a subject orpatient in need thereof.

“Antibody molecule”—As used herein, the term “antibody molecule” isintended to encompass full-length antibodies and antigen bindingfragments thereof, including variants such as modified antibodies,humanized antibodies, germlined antibodies and antigen binding fragmentsthereof. The term “antibody” typically refers to an immunoglobulinpolypeptide having a combination of two heavy and two light chainswherein the polypeptide has significant specific immunoreactive activityto an antigen of interest (herein CD70 or a leukemic stem cell target,for example TIM-3, CD47, IL1RAP). For antibodies of the IgG class, theantibodies comprise two identical light polypeptide chains of molecularweight approximately 23,000 Daltons, and two identical heavy chains ofmolecular weight 53,000-70,000. The four chains are joined by disulfidebonds in a “Y” configuration wherein the light chains bracket the heavychains starting at the mouth of the “Y” and continuing through thevariable region. The light chains of an antibody are classified aseither kappa or lambda (κ,λ). Each heavy chain class may be bound witheither a kappa or lambda light chain. In general, the light and heavychains are covalently bonded to each other, and the “tail” portions ofthe two heavy chains are bonded to each other by covalent disulfidelinkages or non-covalent linkages when the immunoglobulins are generatedeither by hybridomas, B cells or genetically engineered host cells. Inthe heavy chain, the amino acid sequences run from an N-terminus at theforked ends of the Y configuration to the C-terminus at the bottom ofeach chain.

Those skilled in the art will appreciate that heavy chains areclassified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, ε), withsome subclasses among them (e.g., γ1-γ4). It is the nature of this chainthat determines the “class” of the antibody as IgG, IgM, IgA, IgD orIgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1,IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are known toconfer functional specialization. The term “antibody molecule” as usedherein encompasses full-length antibodies or antigen binding fragmentsthereof from any class or subclass of antibody.

With respect to antigen binding fragments encompassed within the genericterm “antibody molecule”, these fragments are parts or portions of afull-length antibody or antibody chain comprising fewer amino acidresidues than an intact or complete antibody whilst retaining antigenbinding activity. The term “antibody molecule” as used herein isintended to encompass antigen binding fragments selected from: anantibody light chain variable domain (VL); an antibody heavy chainvariable domain (VH); a single chain antibody (scFv); a F(ab′)2fragment; a Fab fragment; an Fd fragment; an Fv fragment; a one-armed(monovalent) antibody; diabodies, triabodies, tetrabodies or anyantigen-binding molecule formed by combination, assembly or conjugationof such antigen binding fragments. The term “antibody molecule” as usedherein is further intended to encompass antibody fragments selected fromthe group consisting of: unibodies; domain antibodies; and nanobodies.Fragments can be obtained, for example, via chemical or enzymatictreatment of an intact or complete antibody or antibody chain or byrecombinant means.

“Specificity” and “Multispecific antibodies”—The antibody molecules foruse in the combination therapies described herein bind to particulartarget antigens. It is preferred that the antibody molecules“specifically bind” to their target antigen, wherein the term“specifically bind” refers to the ability of any antibody molecule topreferentially immunoreact with a given target e.g. CD70, TIM-3, CD47,IL1RAP, LILRB2. The antibody molecules of the present combinations andmethods may be monospecific and contain one or more binding sites whichspecifically bind a particular target. The antibody molecules of thepresent combinations and methods may be incorporated into “multispecificantibody” formats, for example bispecific antibodies, wherein themultispecific antibody binds to two or more target antigens. Forexample, in one embodiment, the combination of the present inventioncomprises a bispecific antibody comprising a first antibody moleculespecifically binding to CD70 and a second antibody molecule specificallybinding to TIM-3. In an alternative embodiment, the combination of thepresent invention comprises a bispecific antibody comprising a firstantibody molecule specifically binding to CD70 and a second antibodymolecule specifically binding to CD47. In a further alternativeembodiment, the combination of the present invention comprises abispecific antibody comprising a first antibody molecule specificallybinding to CD70 and a second antibody molecule specifically binding toIL1RAP. In order to achieve multiple specificities, “multispecificantibodies” are typically engineered to include different combinationsor pairings of heavy and light chain polypeptides with different VH-VLpairs. Multispecific, notably bispecific, antibodies may be engineeredso as to adopt the overall conformation of a native antibody, forexample a Y-shaped antibody having Fab arms of different specificitiesconjugated to an Fc region. Alternatively multispecific antibodies, forexample bispecific antibodies, may be engineered so as to adopt anon-native conformation, for example wherein the variable domains orvariable domain pairs having different specificities are positioned atopposite ends of the Fc region.

The bispecific or multispecific antibodies may have a native IgGstructure with two Fab arms having binding specificity for the firsttarget, and one or more additional antigen-binding domains positioned atthe C terminus of the Fc domain having binding specificity for thesecond target. Alternatively, the bispecific or multispecific antibodiesmay have a native IgG structure with two Fab arms having bindingspecificity for the first target and one or more scFv fragments havingbinding specificity for the second target positioned at the C-terminusof the Fc domain. The bispecific or multispecific antibodies may beasymmetric IgG antibodies, such that one Fab region is replaced by adifferent antigen-binding domain, for example a VHH domain. In theseasymmetric antibodies, the Fab region or fragment may bind to CD70 andthe VHH domain may bind to the LSC target or vice versa.

“Modified antibody”—As used herein, the term “modified antibody”includes synthetic forms of antibodies which are altered such that theyare not naturally occurring, e.g., antibodies that comprise at least twoheavy chain portions but not two complete heavy chains (such as, domaindeleted antibodies or minibodies); multispecific forms of antibodies(e.g., bispecific, trispecific, etc.) altered to bind to two or moredifferent antigens or to different epitopes on a single antigen); heavychain molecules joined to scFv molecules and the like. scFv moleculesare known in the art and are described, e.g., in U.S. Pat. 5,892,019. Inaddition, the term “modified antibody” includes multivalent forms ofantibodies (e.g., trivalent, tetravalent, etc., antibodies that bind tothree or more copies of the same antigen). In another embodiment, amodified antibody of the invention is a fusion protein comprising atleast one heavy chain portion lacking a CH2 domain and comprising abinding domain of a polypeptide comprising the binding portion of onemember of a receptor ligand pair.

“Humanizing substitutions”—As used herein, the term “humanizingsubstitutions” refers to amino acid substitutions in which the aminoacid residue present at a particular position in the VH or VL domain ofan antibody is replaced with an amino acid residue which occurs at anequivalent position in a reference human VH or VL domain. The referencehuman VH or VL domain may be a VH or VL domain encoded by the humangermline. Humanizing substitutions may be made in the framework regionsand/or the CDRs of the antibodies, defined herein.

“Humanized variants”—As used herein the term “humanized variant” or“humanized antibody” refers to a variant antibody which contains one ormore “humanizing substitutions” compared to a reference antibody,wherein a portion of the reference antibody (e.g. the VH domain and/orthe VL domain or parts thereof containing at least one CDR) has an aminoacid derived from a non-human species, and the “humanizingsubstitutions” occur within the amino acid sequence derived from anon-human species.

“Germlined variants”—The term “germlined variant” or “germlinedantibody” is used herein to refer specifically to “humanized variants”in which the “humanizing substitutions” result in replacement of one ormore amino acid residues present at (a) particular position(s) in the VHor VL domain of an antibody with an amino acid residue which occurs atan equivalent position in a reference human VH or VL domain encoded bythe human germline. It is typical that for any given “germlinedvariant”, the replacement amino acid residues substituted into thegermlined variant are taken exclusively, or predominantly, from a singlehuman germline-encoded VH or VL domain. The terms “humanized variant”and “germlined variant” are often used interchangeably. Introduction ofone or more “humanizing substitutions” into a camelid-derived (e.g.llama-derived) VH or VL domain results in production of a “humanizedvariant” of the camelid (llama-derived) VH or VL domain. If the aminoacid residues substituted in are derived predominantly or exclusivelyfrom a single human germline-encoded VH or VL domain sequence, then theresult may be a “human germlined variant” of the camelid (llama)-derivedVH or VL domain.

“CD70”—As used herein, the terms “CD70” or “CD70 protein” or “CD70antigen” are used interchangeably and refer to a member of the TNFligand family which is a ligand for TNFRSF7/CD27. CD70 is also known asCD27L or TNFSF7. The terms “human CD70 protein” or “human CD70 antigen”or “human CD70” are used interchangeably to refer specifically to thehuman homolog, including the native human CD70 protein naturallyexpressed in the human body and/or on the surface of cultured human celllines, as well as recombinant forms and fragments thereof. Specificexamples of human CD70 include the polypeptide having the amino acidsequence shown under NCBI Reference Sequence Accession No. NP_001243, orthe extracellular domain thereof.

“Leukemic stem cell target”—As used herein, the term “leukemic stem celltarget” refers to an antigen expressed by leukemic stem cells. Leukemicstem cells or “LSCs” are a low-frequency subpopulation of leukemia cellsthat possess stem cell properties distinct from the bulk leukemia cells,including self-renewal capacity; see for example Wang et al. (2017)Molecular Cancer 16:2, incorporated herein by reference. LSCs typicallyoccur with a frequency in the range of 1 in 10,000 to 1 in 1 million asa proportion of primary blast cells in acute myeloid leukemia—AML(Pollyea and Jordan (2017) Blood 129:1627-1635, incorporated herein byreference). LSCs, if transplanted into an immune-deficient recipient arecapable of initiating leukemic disease and since these leukemic stemcells appear to drive cancer growth, they represent an attractive targetfor novel therapeutic agents. LSCs produce a range of molecules,including cell surface antigens, which serve as markers of LSCs. Thesemarkers may, in some cases, allow LSCs to be distinguished from bulkleukemia cells; see for example Hanekamp et al. (2017) Int. J Hematol.105:549-557, incorporated herein by reference. The term “leukemic stemcell target” as used herein refers to LSC markers, particularly thecell-surface population thereof.

“TIM-3”—As used herein, the term “TIM-3” or “TIMD-3” refers to “T cellimmunoglobulin and mucin-domain containing-3” protein. TIM-3 is alsoreferred to as Hepatitis A virus cellular receptor 2 (HAVCR2). TIM-3 isa member of the immunoglobulin superfamily having a transmembranestructure comprising an extracellular domain consisting of amembrane-distal N-terminal immunoglobulin domain and a membrane-proximalmucin domain containing potential sites for O-linked glycosylation.Different polymorphic variants of the TIM-3 protein are known. See forexample the 301 and 272 amino acid human TIM-3 variants:uniprot.org/uniprot/Q8TDQ0; and uniprot.org/uniprot/E5RHN3. The term“TIM-3” as used herein is intended to cover all TIM-3 polymorphicvariants encoded by transcripts from the TIM-3/HAVCR2 genomic locuswhich result in cell surface-expressed TIM-3.

“Galectin-9”—As used herein, the term “Galectin-9” refers to theextracellular membrane associated protein that serves as a TIM-3 ligandor binding partner. Galectin-9 is a soluble protein containing twotandemly linked carbohydrate recognition domains, which specificallyrecognize the structure of N-linked sugar chains in the TIM-3immunoglobulin domain. The human homolog of Galectin-9 consists of 355amino acid residues as represented by GenBank Accession BAB83625.

“CD47”—As used herein, the term “CD47” refers to the cell surfaceantigen CD47, which is a transmembrane protein ubiquitously expressed ona variety of normal cells and tumor cells. CD47 is a ligand for theimmunoglobulin superfamily receptor SIRPα. CD47 is also referred to as“Antigenic surface determinant protein OA3”, “Integrin-associatedprotein (IAP)” and “Protein MER6”. The human homolog of CD47 encoded bythe CD47 genomic locus is 323 amino acids in length(uniprot.org/uniprot/Q08722). The term CD47 as used herein is intendedto encompass all polymorphic variants of the CD47 protein.

“SIRPα”—As used herein, the term “SIRPα” refers to “Signal-regulatoryprotein alpha”, which is also known as SHP substrate 1 (SHPS-1), BrainIg-like molecule with tyrosine-based activation motifs (Bit), CD172antigen-like family member A, Inhibitory receptor SHPS-1, Macrophagefusion receptor, MyD-1 antigen, SIRPα1, SIRPα2, SIRPα3, p84, and CD172a.SIRPα is a member of the immunoglobulin superfamily and is atransmembrane protein expressed on phagocytic cells, includingmacrophages and dendritic cells. It is a receptor for CD47. The humanhomolog of SIRPα encoded by the SIRPA genomic locus is 504 amino acidsin length (uniprot.org/uniprot/P78324). The term SIRPα as used herein isintended to encompass all polymorphic variants of the SIRPα protein.

“SIRPα antibody molecule fusion protein”—As used herein, the term “SIRPαantibody molecule fusion protein” is intended to mean a fusion proteincomprising the SIRPα protein or a fragment thereof and an antibodymolecule. The antibody molecule may be a full-length antibody moleculeas defined elsewhere herein, for example a full-length IgG antibody.Alternatively, the antibody molecule may be an antigen binding fragmentof an antibody as defined elsewhere herein. The SIRPα protein orfragment thereof may be fused to the antibody molecule at any suitablelocation on the antibody molecule. For example, the SIRPα protein orfragment thereof may be fused to the N-terminus or C-terminus of theheavy chain or light chain of the antibody molecule. In certainembodiments, the SIRPα antibody molecule fusion protein will not includethe full-length SIRPα protein but will include a fragment thereof,particularly a fragment capable of binding to CD47. For example, theSIRPα antibody molecule fusion protein may include one or more copies ofthe SIRPα immunoglobulin V-like domain, wherein the immunoglobulinV-like domain is defined by amino acid positions 32-137 of the 504 aminoacid full-length human SIRPα protein.

“IL1RAP”—As used herein, the term “IL1RAP” or “IL-1RAP” or “IL-1RAcP” or“IL-1RAcP” refers to “Interleukin 1 receptor accessory protein”. IL1RAPis also known as “Interleukin 1 receptor 3” or “IL-1R3” or “IL1R3”.IL1RAP is a co-receptor for type I interleukin 1 receptor (IL1R1) and isrequired for signaling mediated by the cytokine IL-1. It also serves asa co-receptor for IL1R4 and IL1R3 to mediate signaling via IL-33 andIL-36, respectively. IL-1, for example, mediates its effects downstreamof the cell-surface IL-1 receptor complex (IL-1+IL1R1+IL1 RAP) viaactivation of different intracellular signaling pathways including theNF-κB pathway. The human homolog of IL1RAP encoded by the IL1RAP genomiclocus is 570 amino acids in length (uniprot.org/uniprot/Q9NPH3).

“LILRB2”—As used herein, the term “LILRB2” refers to “Leukocyteimmunoglobulin-like receptor subfamily B member 2”. LILRB2 is also knownas “Leukocyte immunoglobulin-like receptor 2” or “LIR-2”, “CD85antigen-like family member D” or “CD85d”, “Immunoglobulin-liketranscript 4” or “ILT-4”, and “Monocyte/macrophage immunoglobulin-likereceptor 10” or “MIR-10”. LILRB2 is involved in the down-regulation ofthe immune response and the development of tolerance. The human homologof LILRB2 encoded by the LILRB2 genomic locus is 598 amino acids inlength (uniprot.org/uniprot/Q8N423).

“Myeloid malignancy”—As used herein, the term “myeloid malignancy”refers to any clonal disease of hematopoietic stem or progenitor cells.Myeloid malignancies or myeloid malignant diseases include chronic andacute conditions. Chronic conditions include myelodysplastic syndromes(MDS), myeloproliferative neoplasms (MPN) and chronic myelomonocyticleukemia (CMML), and acute conditions include acute myeloid leukemia(AML).

“Acute myeloid malignancy”—As used herein, “acute myeloid leukemia” or“AML” refers to hematopoietic neoplasms involving myeloid cells. AML ischaracterized by clonal proliferation of myeloid precursors with reduceddifferentiation capacity. AML patients exhibit an accumulation of blastcells in the bone marrow. “Blast cells”, or simply “blasts”, as usedherein refers to clonal myeloid progenitor cells exhibiting disrupteddifferentiation potential. Blast cells typically also accumulate in theperipheral blood of AML patients. Typically AML is diagnosed if thepatient exhibits 20% or more blast cells in the bone marrow orperipheral blood.

“Anti-cancer agent”—As used herein, an anti-cancer agent refers to anyagent that is capable of preventing, inhibiting or treating cancergrowth directly or indirectly. Such agents include chemotherapeuticagents, immunotherapeutic agents, anti-angiogenic agents, radionuclides,etc., many examples of which are known to those skilled in the art.

“Identity” or “Identical”—As used herein, the term “identity” or“identical”, when used in a relationship between the sequences of two ormore polypeptides, refers to the degree of sequence relatedness betweenpolypeptides, as determined by the number of matches between strings oftwo or more amino acid residues. “Identity” measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by a particular mathematical model orcomputer program (i.e., “algorithms”). Identity of related polypeptidescan be readily calculated by known methods. Such methods include, butare not limited to, those described in Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987;Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M.Stockton Press, New York, 1991; and Carillo et al. (1988) SIAM J.Applied Math. 48:1073. Preferred methods for determining identity aredesigned to give the largest match between the sequences tested. Methodsof determining identity are described in publicly available computerprograms. Preferred computer program methods for determining identitybetween two sequences include the GCG program package, including GAP(Devereux et al. (1984) Nucleic Acids Res. 12:387-395; Genetics ComputerGroup, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, andFASTA (Altschul et al. (1990) J. Mol. Biol. 215:403-410). The BLASTXprogram is publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well-knownSmith Waterman algorithm may also be used to determine identity.

B. Combination Therapy with Anti-CD70 and Anti-LSC Target Antibodies

The present invention relates to combination therapies and their use inthe treatment of malignancies, particularly myeloid malignancies,preferably acute myeloid leukemia (AML). The combinations or combinationtherapies described herein are based on the use of antibody moleculesthat bind CD70 in combination with other agents.

In a first aspect, the combinations or combination therapies of theinvention comprise or consist of antibody molecules that bind todifferent targets. The combinations comprise an antibody molecule thatbinds to CD70 and at least one antibody molecule that binds to aleukemic stem cell target.

All of the combinations in accordance with the present inventioncomprise an antibody molecule that binds to CD70. As described elsewhereherein, CD70 is a member of the tumor necrosis family (TNF) superfamilyof proteins and is a type II transmembrane glycoprotein. It is a ligandfor the TNF receptor CD27. CD70 is transiently expressed onantigen-activated T and B cells and mature dendritic cells and theCD70-CD27 signaling pathway plays an important role in regulating theimmune response.

Constitutive expression of CD70 has been observed on many types ofhematological malignancies and solid carcinomas, rendering this proteinan attractive target for the development of anti-cancer therapies. CD70has been identified as a particularly interesting target for thedevelopment of treatments for myeloid malignancies, specifically acutemyeloid leukemia (AML); see for example Perna et al. (2017) Cancer Cell32:506-519 and Riether et al. (2017) J Exp Med. 214(2):359-380, bothincorporated herein by reference.

CD70 is thought to promote cancer progression in a number of waysincluding direct effects in promoting tumor cell proliferation andsurvival. Upregulated CD70 expression is also thought to play a role inimmunosuppression in the tumor microenvironment by activating Tregulatory cells and dampening the activity of tumor infiltratinglymphocytes (TILs). Based on this tumor immunosuppressive activity, CD70can be classified as an immune checkpoint target. In AML, the expressionof CD70 and its receptor CD27 has been detected on AML blasts, andsignaling via the CD70-CD27 pathway has been linked to the stem-cellbehavior of AML blast populations (Riether et al. (2017) ibid).

In the first aspect of the present invention, the antibody molecule thatbinds CD70 is combined with at least one antibody molecule that binds toa leukemic stem cell target. The combinations may comprise or consist ofan antibody molecule that binds CD70 together with antibody moleculesthat bind to at least two different leukemic stem cell targets, at leastthree different leukemic stem cell targets, at least four differentleukemic stem cell targets, or at least five different leukemic stemcell targets.

Leukemic stem cells or “LSCs” are a distinct population of leukemiacells that possess stem-like properties, for example self-renewalcapacity. In particular embodiments, the LSC targets to which theantibody molecules of the combination bind are independently selectedfrom the group consisting of: TIM-3; Galectin-9; CD47; IL1RAP; LILRB2;CLL-1; CD123; CD33; SAIL; GPR56; CD44; E-selectin; CXCR4; CD25; CD32;PR1; WT1; ADGRE2; CCR1; TNFRSF1B and CD96 (Al-Mawali. (2013) J Stem CellRes Ther. 3(4):1-8; Daria et al. (2016) Leukemia 30:1734-1741; Rashildi& Walter (2016) Expert Review of Hematology 9(4):335-350; Cho et al.(2017) Korean J Intern Med. 32(2):248-257)).

In certain embodiments, the combination comprises or consists of anantibody molecule that binds CD70 and an antibody molecule that binds toa LSC target selected from: TIM-3 or Galectin-9. In preferredembodiments the LSC target is TIM-3.

TIM-3 is a receptor expressed on IFN-γ producing T cells, FoxP3+ Tregcells and innate immune cells (macrophages and dendritic cells). Similarto CD70, TIM-3 has also been classified as an immune checkpoint targetin cancer since the interaction of TIM-3 with its ligands plays animportant role in inhibiting Th1 responses (Das et al. (2017) ImmunolRev. 276(1): 97-111). In the development of cancer, high levels of TIM-3expression have been found to correlate with suppression of T cellsresponses and T cell dysfunction, indicating an important role for TIM-3in dampening the body's anti-tumor immune response (Japp et al. (2015)Cancer Immunol Immunother. 64:1487-1494). In support of this, inhibitionof TIM-3 signaling in preclinical tumor models has been found to restoreanti-tumor immunity (Sakuishi et al. (2010) J Exp Med. 207:2187-2194).TIM-3 has also been identified as a promising therapeutic targetexpressed directly on the surface of leukemic stem cells, particularlyAML stem cells (Jan et al. (2011) Proc Natl Acad Sci. USA 108:5009-5014;Kikushige et al. (2010) Cell Stem Cell. 7:708-717; Kikushige & Miyamoto(2013) Int J Hematol. 98:627-633; Goncalves Silva et al. (2015)Oncotarget 6:33823-33833; Kikushige et al. (2015) Cell Stem Cell17:341-352).

Without wishing to be bound by theory, combination therapies of thepresent invention including antibody molecules that bind to CD70 andantibody molecules that bind to TIM-3 are thought to be particularlyeffective for the treatment of malignancies, particularly myeloidmalignancies, by virtue of the combined effect at the level of theleukemic stem cells. A large body of evidence suggests that LSCs arecritical for the initiation and maintenance of leukemia. Therefore thetargeting of this cell population via CD70 antibodies and antibodiesthat specifically bind a second LSC target such as TIM-3 is thought tobe an effective way in which to target a critical population of tumorcells. CD70 and the LSC targets described herein, particularly TIM-3,also serve as important regulators of the anti-tumor response i.e. theyrepresent immune checkpoint proteins that can be targeted so as tostimulate the body's anti-tumor response. It follows that thecombination therapies described herein are capable of mediating directtherapeutic effects at the level of tumor cells, particularly myeloidleukemia cells, and also indirect effects via stimulation of ananti-tumor immune response.

In certain embodiments, the combination comprises or consists of anantibody molecule that binds to CD70 and an antibody molecule that bindsto the LSC target CD47.

CD47 is a transmembrane protein that displays a relatively ubiquitousexpression pattern. CD47 binds to its receptor SIRPα expressed onphagocytic cells, and this binding interaction transmits a “don't eatme” signal that inhibits phagocytosis of the CD47-expressing cell.Similar to both CD70 and TIM-3, CD47 has been classified as an importantimmune checkpoint target in cancer since the interaction between CD47 ontumor cells and its receptor SIRPα on phagocytic cells has beenidentified as a means by which tumor cells evade phagocytosis mediatedby macrophages, neutrophils and dendritic cells present in the tumorenvironment. CD47 has been found to be highly expressed on a variety ofdifferent tumor cell types, including AML cells (Ponce et al. (2017)Oncotarget 8(7): 11284-11301), and disruption of CD47-SIRPα signalingusing a SIRPα-Fc fusion was found to eliminate AML stem cells in axenograft model (Theocharides et al. (2012) J. Exp. Med. 209(10):1883-1899).

Without wishing to be bound by theory, combination therapies of thepresent invention including antibody molecules that bind CD70 andantibody molecules that bind CD47 are thought to be particularlyeffective for the treatment of malignancies, particularly myeloidmalignancies, by virtue of the combined effect at the level of leukemicstem cells and the innate immune system. The antibody molecule thatbinds to CD70 may serve as an opsonizing antibody and the antibodymolecule that binds to CD47 may enhance phagocytosis of theCD70-expressing tumor cells by blocking the interaction between CD47 andSIRPα.

In certain embodiments, the combination comprises or consists of anantibody molecule that binds to CD70 and an antibody molecule that bindsto the LSC target IL1RAP.

IL1RAP is an immunoglobulin superfamily receptor expressed in liver,skin, placenta, thymus and lung. It serves as a co-receptor for IL1R1 tomediate signaling via the cytokine IL-1, and as a co-receptor for IL1R4and IL1R3 to mediate signaling via IL-33 and IL-36, respectively.Overexpression of IL1RAP has been detected on candidate chronic myeloidleukemia stem cells, and mononuclear cells of patients with acutemyeloid leukemia. Furthermore, antibodies targeting IL1RAP have beenreported as having beneficial therapeutic effects in xenograft models ofCML and AML (Agerstam et al. (2015) Proc Natl Acad Sci USA 112(34):10786-91; Agerstam et al. (2016) Blood 128(23): 2683-2693).

Without wishing to be bound by theory, combination therapies of thepresent invention including antibody molecules that bind to CD70 andantibody molecules that bind to IL1RAP are thought to be particularlyeffective for the treatment of malignancies, particularly myeloidmalignancies, by virtue of the combined effect at the level of leukemicstem cells. Antibodies targeting IL1RAP have been found to beparticularly effective for the killing of CML and AML stem cells (Jaraset al. (2010) Proc Natl Acad Sci USA 107(37): 16280-16285; Askmyr et al.(2013) Blood 121(18):3709-3713). Furthermore, the IL-1 receptor complexis known to signal via the NF-κB signaling pathway and this pathway hasalready been identified as an attractive target in the treatment of AML(see Bosman et al. (2016) Crit Rev Oncol Hematol. 98: 35-44). Itfollows, that the combination of an antibody molecule that binds to CD70and an antibody molecule that binds to IL1RAP may be particularlyefficacious based on dual targeting/inhibition of the NF-κB signalingpathway in LSCs.

In certain embodiments, the combination comprises or consists of anantibody molecule that binds to CD70 and an antibody molecule that bindsto the LSC target LILRB2.

LILRB2 is an immunoglobulin superfamily receptor expressed on a varietyof immune cell types including hematopoietic stem cells, monocytes,macrophages, and dendritic cells. LILRB2 has been implicated in cancerdevelopment, and expression has been reported on various cancer cellsincluding AML and CML cells (Kang et al. (2015) Nat Cell Biol.17:665-677; Colovai et al. (2007) Cytometry B Clin Cytom. 72:354-62).

Without wishing to be bound by theory, combination therapies of thepresent invention including antibody molecules that bind to CD70 andantibody molecules that bind to LILRB2 are thought to be particularlyeffective for the treatment of malignancies, particularly myeloidmalignancies, by virtue of the combined effect at the level of leukemicstem cells. Since LILRB2 has also been identified as a protein thatinteracts with TIM-3, the effect of LILRB2 antibodies may also bemediated via the TIM-3 signaling pathway.

In certain embodiments, the combination comprises or consists of anantibody molecule that binds CD70, an antibody molecule that binds to afirst leukemic stem cell target and an antibody molecule that binds to asecond leukemic stem cell target, wherein the first and second leukemicstem cell targets are different. The first and second leukemic stem celltargets may be independently selected from the group consisting of:TIM-3; Galectin-9; CD47; IL1RAP; LILRB2; CLL-1; CD123; CD33; SAIL;GPR56; CD44; E-selectin; CXCR4; CD25; CD32; PR1; WT1; ADGRE2; CCR1;TNFRSF1B and CD96. In preferred embodiments, the first and secondleukemic stem cell targets are independently selected from the groupconsisting of: TIM-3; Galectin-9; CD47; IL1RAP and LILRB2.

In preferred embodiments, the combination comprises or consists of anantibody molecule that binds CD70, an antibody molecule that binds TIM-3and an antibody molecule that binds CD47. In further preferredembodiments, the combination comprises or consists of an antibodymolecule that binds CD70, an antibody molecule that binds TIM-3 and anantibody molecule that binds IL1RAP. In further preferred embodiments,the combination comprises or consists of an antibody molecule that bindsCD70, an antibody molecule that binds TIM-3, an antibody molecule thatbinds CD47 and an antibody molecule that binds IL1RAP.

The antibody molecules of the present combinations i.e. the antibodymolecules that bind CD70 and the antibody molecules that bind the one ormore LSC targets may be selected from any suitable antibody moleculesdisplaying immunoreactivity for their respective target. As noted above,the term “antibody molecule” is used herein to mean full-lengthantibodies in addition to antigen binding fragments thereof.

The antibodies of the combinations described herein are intended forhuman therapeutic use and therefore, will typically be of the IgA, IgD,IgE, IgG, IgM type, often of the IgG type, in which case they can belongto any of the four sub-classes IgG1, IgG2a and b, IgG3 or IgG4. Inpreferred embodiments, the antibodies of the combinations describedherein are IgG antibodies, preferably IgG1 antibodies.

The antibodies may be monoclonal, polyclonal, multispecific (e.g.bispecific) antibodies, provided that they exhibit the appropriateimmunological specificity for their target(s). Monoclonal antibodies arepreferred since they are highly specific, being directed against asingle antigenic site.

The antigen binding fragments of the combinations described herein willtypically comprise a portion of a full-length antibody, generally theantigen binding or variable domain thereof. Examples of antibodyfragments include Fab, Fab′, F(ab′)2, bi-specific Fab's, and Fvfragments, linear antibodies, single-chain antibody molecules, a singlechain variable fragment (scFv) and multispecific antibodies formed fromantibody fragments (see Holliger and Hudson (2005) Nature Biotechnol.23:1126-36, incorporated herein by reference).

The antibody molecules of the combinations described herein may exhibithigh human homology. Such antibody molecules having high human homologymay include antibodies comprising VH and VL domains of native non-humanantibodies which exhibit sufficiently high % sequence identity to humangermline sequences. In certain embodiments, the antibody molecules arehumanized or germlined variants of non-human antibodies, for exampleantibodies comprising VH and VL domains of camelid conventionalantibodies engineered so as to be humanized, or germlined variants ofthe original antibodies.

In non-limiting embodiments, the antibody molecules of the combinationsmay comprise CH1 domains and/or CL domains (from the heavy chain andlight chain, respectively), the amino acid sequence of which is fully orsubstantially human. For antibody molecules intended for humantherapeutic use, it is typical for the entire constant region of theantibody, or at least a part thereof, to have fully or substantiallyhuman amino acid sequence. Therefore, one or more or any combination ofthe CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (andCH4 domain if present) may be fully or substantially human with respectto its amino acid sequence.

Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain andCL domain (and CH4 domain if present) may all have fully orsubstantially human amino acid sequence. In the context of the constantregion of a humanized or chimeric antibody, or an antibody fragment, theterm “substantially human” refers to an amino acid sequence identity ofat least 90%, or at least 92%, or at least 95%, or at least 97%, or atleast 99% with a human constant region. The term “human amino acidsequence” in this context refers to an amino acid sequence which isencoded by a human immunoglobulin gene, which includes germline,rearranged and somatically mutated genes. The invention alsocontemplates polypeptides comprising constant domains of “human”sequence which have been altered, by one or more amino acid additions,deletions or substitutions with respect to the human sequence, exceptingthose embodiments where the presence of a “fully human” hinge region isexpressly required.

The antibody molecules of the combinations may have one or more aminoacid substitutions, insertions or deletions within the constant regionof the heavy and/or the light chain, particularly within the Fc region.Amino acid substitutions may result in replacement of the substitutedamino acid with a different naturally occurring amino acid, or with anon-natural or modified amino acid. Other structural modifications arealso permitted, such as for example changes in glycosylation pattern(e.g. by addition or deletion of N- or O-linked glycosylation sites).

The antibody molecules of the combinations may be modified with respectto their binding properties to Fc receptors, for example to modulateeffector function. For example cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al. (1992) J. Exp. Med. 176:1191-1195 and Shopes, B (1992) J.Immunol. 148:2918-2922, incorporated herein by reference.

The antibody molecules may also be modified so as to formimmunoconjugates comprising an antibody conjugated to a cytotoxic agentsuch as a chemotherapeutic agent, toxin (e.g., an enzymatically activetoxin of bacterial, fungal, plant or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate). Fc regionsmay also be engineered for half-life extension, for example as describedby Chan and Carter (2010) Nature Reviews: Immunology 10:301-316,incorporated herein by reference.

In yet another embodiment, the Fc region is modified to increase theability of the antibody molecule to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids.

In particular embodiments, the Fc region may be engineered such thatthere is no effector function. In certain embodiments, the antibodymolecules of the invention may have an Fc region derived fromnaturally-occurring IgG isotypes having reduced effector function, forexample IgG4. Fc regions derived from IgG4 may be further modified toincrease therapeutic utility, for example by the introduction ofmodifications that minimize the exchange of arms between IgG4 moleculesin vivo. Fc regions derived from IgG4 may be modified to include theS228P substitution.

In certain embodiments, the antibody molecules of the combinations aremodified with respect to glycosylation. For example, an aglycosylatedantibody can be made (i.e., the antibody lacks glycosylation).Glycosylation can be altered to, for example, increase the affinity ofthe antibody for the target antigen. Such carbohydrate modifications canbe accomplished by, for example, altering one or more sites ofglycosylation within the antibody sequence. For example, one or moreamino acid substitutions can be made that result in elimination of oneor more variable region framework glycosylation sites to therebyeliminate glycosylation at that site. Such aglycosylation may increasethe affinity of the antibody for antigen.

In certain embodiments, the antibody molecules of the combinations arealtered so as to be hypofucosylated i.e. having reduced amounts offucosyl residues, or to be fully or partially de-fucosylated (asdescribed by Natsume et al. (2009) Drug Design Development and Therapy3:7-16) or to have increased bisecting GlcNac structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCactivity of antibodies, producing typically 10-fold enhancement of ADCCrelative to an equivalent antibody comprising a “native” human Fcdomain. Such carbohydrate modifications can be accomplished by, forexample, expressing the antibody in a host cell with alteredglycosylation enzymatic machinery (as described by Yamane-Ohnuki andSatoh (2009) mAbs 1(3):230-236). Examples of non-fucosylated antibodieswith enhanced ADCC function are those produced using the Potelligent™technology of BioWa Inc.

Antibody molecules of the combinations described herein may possessantibody effector function, for example one or more ofantibody-dependent cellular cytotoxicity (ADCC), complement-dependentcytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP).

The antibody molecules of the combinations may be modified within the Fcregion to increase binding affinity for the neonatal Fc receptor FcRn.The increased binding affinity may be measurable at acidic pH (forexample from about approximately pH 5.5 to approximately pH 6.0). Theincreased binding affinity may also be measurable at neutral pH (forexample from approximately pH 6.9 to approximately pH 7.4). By“increased binding affinity” is meant increased binding affinity to FcRnrelative to the unmodified Fc region. Typically the unmodified Fc regionwill possess the wild-type amino acid sequence of human IgG1, IgG2, IgG3or IgG4. In such embodiments, the increased FcRn binding affinity of theantibody molecule having the modified Fc region will be measuredrelative to the binding affinity of wild-type IgG1, IgG2, IgG3 or IgG4for FcRn.

In preferred embodiments, one or more amino acid residues within the Fcregion may be substituted with a different amino acid so as to increasebinding to FcRn. Several Fc substitutions have been reported thatincrease FcRn binding and thereby improve antibody pharmacokinetics.Such substitutions are reported in, for example, Zalevsky et al. (2010)Nat. Biotechnol. 28(2):157-9; Hinton et al. (2006) J Immunol.176:346-356; Yeung et al. (2009) J Immunol. 182:7663-7671; Presta LG.(2008) Curr. Op. Immunol. 20:460-470; and Vaccaro et al. (2005) Nat.Biotechnol. 23(10):1283-88, the contents of which are incorporatedherein by reference in their entirety.

In preferred embodiments, one or more of the antibody molecules of thecombinations described herein comprises a modified human IgG Fc domaincomprising or consisting of the amino acid substitutions H433K andN434F, wherein the Fc domain numbering is in accordance with EUnumbering. In a further preferred embodiment, one or more of theantibody molecules of the combinations described herein comprises amodified human IgG Fc domain comprising or consisting of the amino acidsubstitutions M252Y, S254T, T256E, H433K and N434F, wherein the Fcdomain numbering is in accordance with EU numbering.

CD70 Antibodies

Antibody molecules binding to CD70 that may be incorporated into any ofthe combinations described herein include but are not limited to: CD70antibodies that inhibit interaction of CD70 with CD27; CD70 antibodiesthat compete with CD27 for CD70 binding; CD70 antibodies that inhibitCD70-induced CD27 signaling; CD70 antibodies that inhibit Tregactivation and/or proliferation; CD70 antibodies that depleteCD70-expressing cells; CD70 antibodies that induce lysis ofCD70-expressing cells; CD70 antibodies that possess ADCC, CDCfunctionality, and/or induce ADCP.

Exemplary CD70 antibodies are ARGX-110 described in WO2012/123586(incorporated herein by reference), SGN-70 (WO2006/113909, andMcEarChern et al. (2008) Clin Cancer Res. 14(23):7763, both incorporatedherein by reference) and those CD70 antibodies described inWO2006/044643 and WO2007/038637 (each incorporated herein by reference).

WO2006/044643 describes CD70 antibodies containing an antibody effectordomain which can mediate one or more of ADCC, ADCP, CDC or ADC andeither exert a cytostatic or cytotoxic effect on a CD70-expressingcancer or exert an immunosuppressive effect on a CD70-expressingimmunological disorder in the absence of conjugation to a cytostatic orcytotoxic agent. The antibodies exemplified therein are based on theantigen-binding regions of two monoclonal antibodies, denoted 1F6 and2F2.

WO2007/038637 describes fully human monoclonal antibodies that bind toCD70. These antibodies are characterized by binding to human CD70 with aK_(D) of 1×10⁻⁷ M or less. The antibodies also bind to, and areinternalized by, renal cell carcinoma tumor cell lines which expressCD70, such as 786-O.

ARGX-110 is an IgG1 anti-CD70 antibody that has been shown to inhibitthe interaction of CD70 with its receptor CD27 (Silence et al. (2014)MAbs. Mar-Apr;6(2):523-32, incorporated herein by reference). Inparticular, ARGX-110 has been shown to inhibit CD70-induced CD27signaling. Levels of CD27 signaling may be determined by, for example,measurement of serum soluble CD27 as described in Riether et al. (supra)or of IL-8 expression as described in Silence et al. (supra). Withoutbeing bound by theory, inhibiting CD27 signaling is thought to reduceactivation and/or proliferation of Treg cells, thereby reducinginhibition of anti-tumor effector T cells. AGRX-110 has also beendemonstrated to deplete CD70-expressing tumor cells. In particular,ARGX-110 has been shown to lyse CD70-expressing tumor cells viaantibody-dependent cellular cytotoxicity (ADCC) and complement-dependentcytotoxicity (CDC), and also to increase antibody-dependent cellularphagocytosis (ADCP) of CD70-expressing cells (Silence et al., supra).

The CDR, VH and VL amino acid sequences of ARGX-110 are shown in thetable below.

TABLE 1  SEQ  ID ARGX-110 Sequence NO: HCDR1 VYYMN 1 HCDR2DINNEGGTTYYADSVKG 2 HCDR3 DAGYSNHVPIFDS 3 VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVR 4QAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGT LVTVSS LCDR1 GLKSGSVTSDNFPT 5LCDR2 NTNTRHS 6 LCDR3 ALFISNPSVE 7 VLQAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSDNFPTWYQ 8QTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLG

In certain embodiments, the antibody molecule that binds to CD70comprises a variable heavy chain domain (VH) and a variable light chaindomain (VL) wherein the VH and VL domains comprise or consist of the CDRsequences:

-   -   HCDR3 comprising or consisting of SEQ ID NO: 3;    -   HCDR2 comprising or consisting of SEQ ID NO: 2;    -   HCDR1 comprising or consisting of SEQ ID NO: 1;    -   LCDR3 comprising or consisting of SEQ ID NO: 7;    -   LCDR2 comprising or consisting of SEQ ID NO: 6; and    -   LCDR1 comprising or consisting of SEQ ID NO: 5.

In certain embodiments, the antibody molecule that binds to CD70comprises a variable heavy chain domain (VH domain) comprising orconsisting of a sequence at least 70%, at least 80%, at least 90% or atleast 95% identical to SEQ ID NO: 4 and a variable light chain domain(VL domain) comprising or consisting of a sequence at least 70%, atleast 80%, at least 90% or at least 95% identical to SEQ ID NO: 8. Incertain embodiments, the antibody molecule that binds to CD70 comprisesa variable heavy chain domain (VH domain) comprising or consisting ofSEQ ID NO: 4 and a variable light chain domain (VL domain) comprising orconsisting of SEQ ID NO: 8.

CD70 antibody molecules that may be incorporated into the combinationsdescribed herein include antibody drug conjugates (ADCs). ADCs areantibodies attached to active agents, for example auristatins andmaytansines or other cytotoxic agents. Certain ADCs maintain antibodyblocking and/or effector function (e.g. ADCC, CDC, ADCP) while alsodelivering the conjugated active agent to cells expressing the target(e.g. CD70). Examples of anti-CD70 ADCs include vorsetuzumab mafodotin(also known as SGN-75, Seattle Genetics), SGN-70A (Seattle Genetics),and MDX-1203/BMS936561 (Bristol-Myers Squibb), each of which may be usedin accordance with the invention. Suitable anti-CD70 ADCs are alsodescribed in WO2008074004 and WO2004073656, each of which isincorporated herein by reference.

CD70 antibody molecules that may be incorporated into the combinationsdescribed herein also include SIRPα-antibody molecule fusion proteins or“licMABs” (local inhibitory checkpoint monoclonal antibodies), asdescribed for example in Ponce et al. (2017) (supra). TheseSIRPα-antibody molecule fusion proteins or licMABs comprise an antibodyor antibody molecule (in this case a CD70 antibody or antibody molecule)fused to a domain of the SIRPα protein, in particular, the extracellularimmunoglobulin V-like domain.

LSC Target Antibodies

TIM-3 and Galectin-9 Antibodies

Antibody molecules binding to LSC targets that may be incorporated intothe combinations described herein include antibody molecules thatmediate their effects via TIM-3. These effects may be mediated viadirect binding to TIM-3 or via binding to a LSC target associated withTIM-3 signaling. In certain embodiments, the LSC target antibodymolecules of the combinations described herein inhibit the interactionof TIM-3 with one or more TIM-3 interacting proteins. The TIM-3interacting proteins may be selected from: CEACAM-1; HMGB-1;phosphatidylserine; Galectin-9; LILRB2; and combinations thereof. In oneembodiment, the LSC target antibody molecule of the combination inhibitsthe interaction of TIM-3 with its ligand, Galectin-9. In one embodiment,the LSC target antibody molecule of the combination inhibits theinteraction of TIM-3 with its ligand, LILRB2.

In certain embodiments, the LSC target antibody molecule of thecombinations binds to Galectin-9. In certain embodiments, the LSC targetantibody molecule of the combinations binds to TIM-3. For embodimentswherein the LSC target is TIM-3, the antibody molecule may achieve oneor more of the following effects: reduced NF-κB signaling; reducedWnt/β-catenin signaling; reduced stemness of AML cells; or a combinationthereof. For antibody molecules that bind to TIM-3, the antibodymolecules may inhibit the interaction of TIM-3 with one or more TIM-3interacting proteins. The TIM-3 interacting proteins may be selectedfrom: CEACAM-1; HMGB-1; phosphatidylserine; Galectin-9; and combinationsthereof. In one embodiment, the antibody molecule that binds to TIM-3inhibits the interaction of TIM-3 with its ligand, Galectin-9.

In certain embodiments, the LSC target antibody molecule of thecombinations inhibits the interaction of TIM-3 and LILRB2. In suchembodiments, the antibody molecule preferably binds TIM-3 and inhibitsbinding of TIM-3 to LILRB2.

Antibody molecules that bind to TIM-3 and that may be incorporated intothe combinations described herein include but are not limited to theTIM-3 antibodies described in any of the following: U.S. Pat. No.8,647,623; 8,552,156; 9,605,070; 8,841,418; 9,631,026; 9,556,270;WO2016/111947, each of which is incorporated herein by reference.Antibody molecules that bind to TIM-3 and that may be incorporated intothe combinations described herein also include but are not limited to:clone F38-2E2; MBG453 (Novartis); ATIK2a (Kyowa Kirin).

Antibody molecules that bind to Galectin-9 and that may be incorporatedinto the combinations described herein include but are not limited toclone 9M1-3.

CD47 Antibodies

In certain embodiments, the combinations of the invention comprise anantibody molecule that binds CD47. CD47 antibody molecules for use inthe combinations described herein are antibody molecules that inhibitthe interaction between CD47 and SIRPα. As noted elsewhere herein, theinteraction between the ligand CD47 expressed by the LSC and thereceptor SIRPα expressed by phagocytic cells transmits a “don't eat me”signal downstream of the SIRPα receptor. The CD47 antibody molecules ofthe combinations described herein can therefore increase phagocytosis oftumor cells, particularly LSCs.

A variety of CD47 antibodies are available, including CD47 antibodies atdifferent stages of clinical development. The skilled person willappreciate that any CD47 antibody suitable for human therapeutic use maybe incorporated into the combinations described herein. Exemplary CD47antibodies include but are not limited to Hu5F9-G4; CC-90002; ALX148 andclone B6H12.2.

IL1RAP Antibodies

In certain embodiments, the combinations of the invention comprise anantibody molecule that binds IL1RAP. IL1RAP antibody molecules for usein the combinations described herein are preferably antibody moleculesthat bind to IL1RAP and inhibit signaling via the IL-1 receptor complexat the cell surface.

IL1RAP antibodies have been described, see Agerstam et al. (2015)(supra), and also WO2012/098407 and WO2014/100772. The skilled personwill appreciate that any IL1RAP antibody suitable for human therapeuticuse may be incorporated into the combinations described herein.

Camelid-Derived LSC Target Antibodies

The antibody molecules specifically binding to LSC targets, particularlyantibody molecules specifically binding to TIM-3, Galectin-9, CD47,IL1RAP and/or LILRB2, may be camelid-derived.

For example, the antibody molecules may be selected from immunelibraries obtained by a method comprising the step of immunizing acamelid with the LSC target of interest. The camelid may be immunizedwith a LSC target protein or polypeptide fragment thereof, or with anmRNA molecule or cDNA molecule expressing the protein or a polypeptidefragment thereof. Methods for producing antibodies in camelid speciesand selecting antibodies against preferred targets from camelid immunelibraries are described in, for example, International PatentApplication No. WO2010/001251, incorporated herein by reference.

In certain embodiments, the antibody molecules may be camelid-derived inthat they comprise at least one hypervariable loop or complementaritydetermining region obtained from a VH domain or a VL domain of a speciesin the family Camelidae. In particular, the antibody molecule maycomprise VH and/or VL domains, or CDRs thereof, obtained by activeimmunization of outbred camelids, e.g. llamas, with TIM-3, Galectin-9,CD47 or IL1RAP for example.

The term “obtained from” in this context implies a structuralrelationship, in the sense that the HVs or CDRs of the antibody moleculeembody an amino acid sequence (or minor variants thereof) which wasoriginally encoded by a Camelidae immunoglobulin gene. However, thisdoes not necessarily imply a particular relationship in terms of theproduction process used to prepare the antibody molecule.

Camelid-derived antibody molecules may be derived from any camelidspecies, including inter alia, llama, dromedary, alpaca, vicuna, guanacoor camel.

Antibody molecules comprising camelid-derived VH and VL domains, or CDRsthereof, are typically recombinantly expressed polypeptides, and may bechimeric polypeptides. The term “chimeric polypeptide” refers to anartificial (non-naturally occurring) polypeptide which is created byjuxtaposition of two or more peptide fragments which do not otherwiseoccur contiguously. Included within this definition are “species”chimeric polypeptides created by juxtaposition of peptide fragmentsencoded by two or more species, e.g. camelid and human.

In certain embodiments, the entire VH domain and/or the entire VL domainmay be obtained from a species in the family Camelidae. In specificembodiments, the camelid-derived VH domain may comprise an amino acidsequence selected from SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 121 and 123, whereas the camelid-derived VLdomain may comprise an amino acid sequence selected from SEQ ID NOs: 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 122 and 124. Thecamelid-derived VH domain and/or the camelid-derived VL domain may thenbe subject to protein engineering, in which one or more amino acidsubstitutions, insertions or deletions are introduced into the camelidamino acid sequence. These engineered changes preferably include aminoacid substitutions relative to the camelid sequence. Such changesinclude “humanization” or “germlining” wherein one or more amino acidresidues in a camelid-encoded VH or VL domain are replaced withequivalent residues from a homologous human-encoded VH or VL domain. Incertain embodiments, the camelid-derived VH domain may exhibit at least90%, 95%, 97%, 98% or 99% identity with the amino acid sequence shown asSEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,121 or 123. Alternatively, or in addition, the camelid-derived VL domainmay exhibit at least 90%, 95%, 97%, 98% or 99% identity with the aminoacid sequence shown as SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 122 or 124.

Isolated camelid VH and VL domains obtained by active immunization of acamelid (e.g. llama) with a LSC target antigen (for example) can be usedas a basis for engineering antibody molecules for use in thecombinations described herein. Starting from intact camelid VH and VLdomains, it is possible to engineer one or more amino acidsubstitutions, insertions or deletions which depart from the startingcamelid sequence. In certain embodiments, such substitutions, insertionsor deletions may be present in the framework regions of the VH domainand/or the VL domain.

In other embodiments, there are provided “chimeric” antibody moleculescomprising camelid-derived VH and VL domains (or engineered variantsthereof) and one or more constant domains from a non-camelid antibody,for example human-encoded constant domains (or engineered variantsthereof). In such embodiments it is preferred that both the VH domainand the VL domain are obtained from the same species of camelid, forexample both VH and VL may be from Lama glama or both VH and VL may befrom Lama pacos (prior to introduction of engineered amino acid sequencevariation). In such embodiments both the VH and the VL domain may bederived from a single animal, particularly a single animal which hasbeen actively immunised with the antigen of interest.

As an alternative to engineering changes in the primary amino acidsequence of Camelidae VH and/or VL domains, individual camelid-derivedhypervariable loops or CDRs, or combinations thereof, can be isolatedfrom camelid VH/VL domains and transferred to an alternative (i.e.non-Camelidae) framework, e.g. a human VH/VL framework, by CDR grafting.

In certain embodiments, the antibody molecules that bind to TIM-3 areselected from antibody molecules comprising a combination of variableheavy chain CDR3 (HCDR3), variable heavy chain CDR2 (HCDR2) and variableheavy chain CDR1 (HCDR1), variable light chain CDR3 (LCDR3), variablelight chain CDR2 (LCDR2) and variable light chain CDR1 (LCDR1) selectedfrom the following:

-   -   (i) HCDR3 comprising SEQ ID NO: 41; HCDR2 comprising SEQ ID NO:        40; HCDR1 comprising SEQ ID NO: 39; LCDR3 comprising SEQ ID NO:        80; LCDR2 comprising SEQ ID NO: 79; and LCDR1 comprising SEQ ID        NO: 78;    -   (ii) HCDR3 comprising SEQ ID NO: 43; HCDR2 comprising SEQ ID NO:        42; HCDR1 comprising SEQ ID NO: 39; LCDR3 comprising SEQ ID NO:        83; LCDR2 comprising SEQ ID NO: 82; and LCDR1 comprising SEQ ID        NO: 81;    -   (iii) HCDR3 comprising SEQ ID NO: 46; HCDR2 comprising SEQ ID        NO: 45; HCDR1 comprising SEQ ID NO: 44; LCDR3 comprising SEQ ID        NO: 86; LCDR2 comprising SEQ ID NO: 85; and LCDR1 comprising SEQ        ID NO: 84;    -   (iv) HCDR3 comprising SEQ ID NO: 49; HCDR2 comprising SEQ ID NO:        48; HCDR1 comprising SEQ ID NO: 47; LCDR3 comprising SEQ ID NO:        88; LCDR2 comprising SEQ ID NO: 82; and LCDR1 comprising SEQ ID        NO: 87;    -   (v) HCDR3 comprising SEQ ID NO: 52; HCDR2 comprising SEQ ID NO:        51; HCDR1 comprising SEQ ID NO: 50; LCDR3 comprising SEQ ID NO:        91; LCDR2 comprising SEQ ID NO: 90; and LCDR1 comprising SEQ ID        NO: 89;    -   (vi) HCDR3 comprising SEQ ID NO: 55; HCDR2 comprising SEQ ID NO:        54; HCDR1 comprising SEQ ID NO: 53; LCDR3 comprising SEQ ID NO:        94; LCDR2 comprising SEQ ID NO: 93; and LCDR1 comprising SEQ ID        NO: 92;    -   (vii) HCDR3 comprising SEQ ID NO: 58; HCDR2 comprising SEQ ID        NO: 57; HCDR1 comprising SEQ ID NO: 56; LCDR3 comprising SEQ ID        NO: 97; LCDR2 comprising SEQ ID NO: 96; and LCDR1 comprising SEQ        ID NO: 95;    -   (viii) HCDR3 comprising SEQ ID NO: 60; HCDR2 comprising SEQ ID        NO: 59; HCDR1 comprising SEQ ID NO: 50; LCDR3 comprising SEQ ID        NO: 100; LCDR2 comprising SEQ ID NO: 99; and LCDR1 comprising        SEQ ID NO: 98;    -   (ix) HCDR3 comprising SEQ ID NO: 63; HCDR2 comprising SEQ ID NO:        62; HCDR1 comprising SEQ ID NO: 61; LCDR3 comprising SEQ ID NO:        103; LCDR2 comprising SEQ ID NO: 102; and LCDR1 comprising SEQ        ID NO: 101;    -   (x) HCDR3 comprising SEQ ID NO: 65; HCDR2 comprising SEQ ID NO:        64; HCDR1 comprising SEQ ID NO: 39; LCDR3 comprising SEQ ID NO:        106; LCDR2 comprising SEQ ID NO: 105; and LCDR1 comprising SEQ        ID NO: 104;    -   (xi) HCDR3 comprising SEQ ID NO: 67; HCDR2 comprising SEQ ID NO:        66; HCDR1 comprising SEQ ID NO: 50; LCDR3 comprising SEQ ID NO:        109; LCDR2 comprising SEQ ID NO: 108; and LCDR1 comprising SEQ        ID NO: 107;    -   (xii) HCDR3 comprising SEQ ID NO: 69; HCDR2 comprising SEQ ID        NO: 68; HCDR1 comprising SEQ ID NO: 50; LCDR3 comprising SEQ ID        NO: 112; LCDR2 comprising SEQ ID NO: 111; and LCDR1 comprising        SEQ ID NO: 110;    -   (xiii) HCDR3 comprising SEQ ID NO: 72; HCDR2 comprising SEQ ID        NO: 71; HCDR1 comprising SEQ ID NO: 70; LCDR3 comprising SEQ ID        NO: 115; LCDR2 comprising SEQ ID NO: 114; and LCDR1 comprising        SEQ ID NO: 113;    -   (xiv) HCDR3 comprising SEQ ID NO: 74; HCDR2 comprising SEQ ID        NO: 73; HCDR1 comprising SEQ ID NO: 50; LCDR3 comprising SEQ ID        NO: 117; LCDR2 comprising SEQ ID NO: 111; and LCDR1 comprising        SEQ ID NO: 116; and    -   (xv) HCDR3 comprising SEQ ID NO: 77; HCDR2 comprising SEQ ID NO:        76; HCDR1 comprising SEQ ID NO: 75; LCDR3 comprising SEQ ID NO:        120; LCDR2 comprising SEQ ID NO: 119; and LCDR1 comprising SEQ        ID NO: 118.

In certain embodiments, the antibody molecules that bind to TIM-3 areselected from antibody molecules comprising or consisting of a variableheavy chain domain (VH) and a variable light chain domain (VL) selectedfrom the following:

-   -   (i) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 9 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 10 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto;    -   (ii) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 11 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 12 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto;    -   (iii) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 13 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 14 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto;    -   (iv) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 15 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 16 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto;    -   (v) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 17 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 18 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto;    -   (vi) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 19 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 20 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto;    -   (vii) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 21 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 22 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto;    -   (viii) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 23 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 24 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto;    -   (ix) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 25 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 26 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto;    -   (x) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 27 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 28 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto;

(xi) a VH domain comprising or consisting of the amino acid sequence ofSEQ ID NO: 29 or an amino acid sequence having at least 80%, 90%, 95%,98% 99% identity thereto, and a VL domain comprising or consisting ofthe amino acid sequence of SEQ ID NO: 30 or an amino acid sequencehaving at least 80%, 90%, 95%, 98% 99% identity thereto;

-   -   (xii) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 31 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 32 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto;    -   (xiii) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 33 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 34 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto;    -   (xiv) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 35 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 36 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto;    -   (xv) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID NO: 37 or an amino acid sequence having at        least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domain        comprising or consisting of the amino acid sequence of SEQ ID        NO: 38 or an amino acid sequence having at least 80%, 90%, 95%,        98% 99% identity thereto.

In certain embodiments, the antibody molecules that bind to IL1RAP areselected from antibody molecules comprising a combination of variableheavy chain CDR3 (HCDR3), variable heavy chain CDR2 (HCDR2) and variableheavy chain CDR1 (HCDR1), variable light chain CDR3 (LCDR3), variablelight chain CDR2 (LCDR2) and variable light chain CDR1 (LCDR1) selectedfrom the following:

-   -   (i) HCDR3 comprising SEQ ID NO: 127; HCDR2 comprising SEQ ID NO:        126;        -   HCDR1 comprising SEQ ID NO: 125; LCDR3 comprising SEQ ID NO:            133;        -   LCDR2 comprising SEQ ID NO: 132; and LCDR1 comprising SEQ ID            NO: 131; and    -   (ii) HCDR3 comprising SEQ ID NO: 130; HCDR2 comprising SEQ ID        NO: 129;        -   HCDR1 comprising SEQ ID NO: 128; LCDR3 comprising SEQ ID NO:            136;        -   LCDR2 comprising SEQ ID NO: 135; and LCDR1 comprising SEQ ID            NO: 134.

In certain embodiments, the antibody molecules that bind to IL1RAP areselected from antibody molecules comprising or consisting of a variableheavy chain domain (VH) and a variable light chain domain (VL) selectedfrom the following:

-   -   (i) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID        -   NO: 121 or an amino acid sequence having at least 80%, 90%,            95%, 98% 99% identity thereto, and a VL domain comprising or            consisting of the amino acid sequence of SEQ ID NO: 122 or            an amino acid sequence having at least 80%, 90%, 95%, 98%            99% identity thereto; and    -   (ii) a VH domain comprising or consisting of the amino acid        sequence of SEQ ID

NO: 123 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99%identity thereto, and a VL domain comprising or consisting of the aminoacid sequence of SEQ ID NO: 124 or an amino acid sequence having atleast 80%, 90%, 95%, 98% 99% identity thereto.

For embodiments wherein the domains of the antibodies or antigen bindingfragments are defined by a particular percentage sequence identity to areference sequence, the VH and/or VL domains may retain identical CDRsequences to those present in the reference sequence such that thevariation is present only within the framework regions.

Antibody molecules comprising camelid-derived VH and VL domains, or CDRsthereof, can take various different antibody forms in which both a VHdomain and a VL domain are present. Antibodies and antigen bindingfragments within the definition of “antibody molecule” as used in thecontext of the claimed combinations are described elsewhere herein.

Formulation of the Combination

The different antibody molecules of the combinations may be combined orformulated in any manner allowing the combination therapy to beadministered to a subject or patient in need thereof, preferably a humansubject or patient. The combination may be formulated for single doseadministration or for multiple dose administration.

For embodiments wherein the antibody molecules are antigen bindingfragments, the antibody molecules may be combined as a multispecificantibody, for example a bispecific antibody. For example, if thecombination comprises a Fab fragment that binds CD70 and a Fab fragmentthat binds to a LSC target, the two Fab fragments may be incorporatedinto a single bispecific antibody molecule having the two Fab regionsconjugated to an IgG Fc portion. In certain embodiments, the combinationcomprises or consists of a multispecific antibody, preferably abispecific antibody, comprising an antibody molecule that binds CD70 andan antibody molecule that binds TIM-3. In certain embodiments, thecombination comprises or consists of a multispecific antibody,preferably a bispecific antibody, comprising an antibody molecule thatbinds CD70 and an antibody molecule that binds CD47. In certainembodiments, the combination comprises or consists of a multispecificantibody, preferably a bispecific antibody, comprising an antibodymolecule that binds CD70 and an antibody molecule that binds IL1RAP.

Bispecific or multispecific antibodies in accordance with the presentinvention may be configured according to any suitablebispecific/multispecific antibody format as described elsewhere herein.For example, the antibody molecules of the combination may beincorporated into a bispecific or multispecific antibody format suchthat the antibody binds to the different targets in “trans”, for examplethe situation where each Fab arm of the Y-shaped antibody has adifferent binding specificity. In alternative embodiments, the antibodymolecules may be incorporated into a bispecific or multispecificantibody format such that the targets are bound in the “cis” position.For example, the Fab regions or variable domains thereof may bepositioned at opposite ends of an IgG Fc portion. In certainembodiments, the antibody molecules may be incorporated in an asymmetricbispecific IgG antibody format wherein the first antibody molecule is aFab fragment forming one arm of the “Y”-shaped antibody and the secondantibody molecule is a VHH domain.

In certain embodiments, antibody molecules of the combination areseparate molecules that are co-formulated i.e. formulated as a singlepharmaceutical composition. For embodiments wherein the antibodymolecules are co-formulated, the combination or composition is suitablefor simultaneous administration of the two components. The compositionmay be formulated for single dose administration or multiple doseadministration. For embodiments in which the antibody molecules areco-formulated, the antibody molecules may be formulated in equivalentamounts, for example according to a 1:1 ratio for a combinationcomprising first and second antibody molecules binding to differenttargets. Alternatively, the antibody molecules may be formulated suchthat the ratio of the different antibody molecules is not 1:1. Forexample, for embodiments wherein the combination comprises or consistsof first and second antibody molecules binding to different targets, theratio of first and second antibody molecules may be 2:1, optionally 3:1,optionally 4:1. Alternatively, the antibody molecules may be formulatedaccording to a ratio of 1:2, optionally 1:3, optionally 1:4.

In certain embodiments, the antibody molecules of the combination areformulated separately, for example as individual compositions. Forembodiments wherein the antibody molecules are formulated separately,the possibility exists for simultaneous or separate administration ofthe different components or compositions. If the antibody molecules orthe separate compositions containing them are administered separately,there may be sequential administration of the antibody molecules orcompositions in either order. For example, the antibody molecule thatbinds to CD70 may be administered first followed by the antibodymolecule that binds to the leukemic stem cell target or vice versa. Theinterval between administration of the antibody molecules orcompositions may be any suitable time interval. The administration ofthe different compositions may be carried out once (for a single doseadministration) or repeatedly (for a multiple dose administration).

For embodiments wherein the antibody molecules are co-formulated and/orfor embodiments wherein the antibody molecules are provided as separatecompositions, the antibody molecules may be formulated using anysuitable pharmaceutical carriers or excipients.

Techniques for formulating antibodies for human therapeutic use are wellknown in the art and are reviewed, for example, in Wang et al. (2007)Journal of Pharmaceutical Sciences, 96:1-26, the contents of which areincorporated herein by reference in their entirety. For embodimentswherein the antibody molecules are formulated separately, thepharmaceutical carriers or excipients may be different for the differentcompositions or the same.

Pharmaceutically acceptable excipients that may be used to formulate thecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (for example sodiumcarboxymethylcellulose), polyethylene glycol, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

In certain embodiments, the compositions are formulated foradministration to a subject via any suitable route of administrationincluding but not limited to intramuscular, intravenous, intradermal,intraperitoneal injection, subcutaneous, epidural, nasal, oral, rectal,topical, inhalational, buccal (e.g., sublingual), and transdermaladministration. For embodiments wherein the antibody molecules areformulated separately, each composition may be formulated foradministration via a different route.

For combinations of the invention comprising or consisting of agents inaddition to the CD70 antibody molecule and the LSC target antibodymolecule, the one or more additional agents may be formulated foradministration via the same route or via a different route as comparedwith the first and second antibody molecules. For example, inembodiments wherein the combination includes an antibody molecule thatbinds to CD70, an antibody molecule that binds to a LSC target andazacitidine, the antibody molecules may be administered intravenouslywhilst the azacitidine may be administered subcutaneously via injection.

C. Combination Therapy with Anti-CD70 Antibodies and SIRPα Inhibitors

In a second aspect, the combinations or combination therapies of theinvention comprise or consist of an antibody molecule that binds to CD70and an agent that inhibits SIRPα signaling. Antibody molecules that bindto CD70 and that are suitable for use in the combinations of the presentinvention are described above and all embodiments presented in thecontext of the first aspect of the invention are equally applicable tothis second aspect.

In the combinations or combination therapies of the second aspect of theinvention, the antibody molecule that binds to CD70 is combined with anagent that inhibits SIRPα signaling. As explained elsewhere herein,SIRPα is a receptor expressed on the surface of phagocytic cellsincluding in particular macrophages, neutrophils and dendritic cells.SIRPα is a receptor for the ligand CD47, and this ligand is expressed onthe surface of a variety of different cell types. The binding of CD47 toSIRPα triggers an intracellular signaling pathway downstream of SIRPαwithin the phagocyte which serves to down-regulate the phagocyticactivity. The consequence of this is that the CD47-SIRPα signaling axispromotes survival of CD47-expressing cells by preventing clearance ofthese cells by the phagocytic cells of the immune system.

As used herein, the term “agent that inhibits SIRPα signaling” isintended to mean any agent that interferes with the CD47-SIRPα signalingaxis such that the “don't eat me” signal generated by this pathway issuppressed. In certain embodiments, the agent that inhibits SIRPαsignaling is an antibody molecule that binds CD47 and inhibits theinteraction between CD47 and SIRPα. In other embodiments, the agent thatinhibits SIRPα signaling is an antibody molecule that binds SIRPα andinhibits the interaction between CD47 and SIRPα. Antibodies that bind toCD47 and SIRPα, respectively, are known in the art and could be includedin the combinations described herein. Exemplary SIRPα antibodiessuitable for use in the combinations described herein include but arenot limited to: clone KWAR23; clone B4B6; and clone OX-119.

In certain embodiments, the agent that inhibits SIRPα signaling is aSIRPα antibody-molecule fusion protein. As defined elsewhere herein,SIRPα antibody-molecule fusion proteins comprise SIRPα or a fragmentthereof together with an antibody or fragment thereof. In certainembodiments, the SIRPα antibody-molecule fusion protein comprises atleast one copy of the immunoglobulin V-like domain of SIRPα, optionallymultiple copies of this immunoglobulin V-like domain of SIRPα.

In certain embodiments, the agent that inhibits SIRPα signalingcomprises SIRPα or the immunoglobulin V-like domain therefrom covalentlylinked to the Fc region of an antibody, for example an IgG1 antibody. Inone embodiment, the agent that inhibits SIRPα signaling is TTI-621(Trillium Therapeutics Inc).

In certain embodiments, the agent that inhibits SIRPα signalingcomprises SIRPα or the immunoglobulin V-like domain therefrom covalentlylinked to a full-length IgG antibody, for example a full-length IgG1antibody.

In preferred embodiments of the combination in accordance with thissecond aspect of the invention, the combination comprises or consists ofan antibody molecule that binds to CD70 wherein the antibody molecule islinked to SIRPα or is linked to at least one copy of the immunoglobulinV-like domain of SIRPα. The linkage is preferably covalent. The CD70antibody molecule may be linked to multiple copies of the immunoglobulinV-like domain of SIRPα, for example two, three, four or more copies. TheCD70 antibody molecule may be linked to the SIRPα domain directly orindirectly via a linker, for example a polyglycine-serine linker.

For embodiments wherein the CD70 antibody molecule is linked, preferablycovalently linked, to at least one copy of the immunoglobulin V-likedomain of SIRPα, the CD70 antibody molecule preferably comprises avariable heavy chain domain (VH) and a variable light chain domain (VL)wherein the VH and VL domains comprise the CDR sequences:

-   -   HCDR3 comprising or consisting of SEQ ID NO: 3;    -   HCDR2 comprising or consisting of SEQ ID NO: 2;    -   HCDR1 comprising or consisting of SEQ ID NO: 1;    -   LCDR3 comprising or consisting of SEQ ID NO: 7;    -   LCDR2 comprising or consisting of SEQ ID NO: 6; and    -   LCDR1 comprising or consisting of SEQ ID NO: 5.

In certain embodiments, the antibody molecule that binds to CD70 andthat is linked to at least one copy of the immunoglobulin V-like domainof SIRPα comprises a variable heavy chain domain (VH domain) comprisingor consisting of a sequence at least 70%, at least 80%, at least 90% orat least 95% identical to SEQ ID NO: 4 and a variable light chain domain(VL domain) comprising or consisting of a sequence at least 70%, atleast 80%, at least 90% or at least 95% identical to SEQ ID NO: 8. Incertain embodiments, the antibody molecule that binds to CD70 and thatis linked to at least one copy of the immunoglobulin V-like domain ofSIRPα comprises a variable heavy chain domain (VH domain) comprising orconsisting of SEQ ID NO: 4 and a variable light chain domain (VL domain)comprising or consisting of SEQ ID NO: 8.

D. Additional Agents

The combinations according to the first and second aspects of theinvention may include, in addition to the antibody molecules and agentsdescribed above, one or more additional anti-cancer agents. For example,the combinations may comprise at least one additional agent for thetreatment of myeloid malignancy, particularly for the treatment of AML.

In certain embodiments, the combinations described herein comprise anucleoside metabolic inhibitor (or NMI). For example, the combinationsmay comprise a hypomethylating agent, for example azacitidine (alsoreferred to herein as azacytidine, AZA or aza) or decitabine.Azacitidine is an analogue of cytidine and decitabine is its deoxyderivative. AZA and decitabine are inhibitors of DNA methyltransferases(DNMT) known to upregulate gene expression by promoter hypomethylation.Such hypomethylation disrupts cell function, thereby resulting incytotoxic effects.

In particular embodiments, the combinations described herein comprise orconsist of an antibody molecule that binds to CD70, an antibody moleculethat binds to TIM-3 and azacitidine. In particular embodiments, thecombinations described herein comprise or consist of an antibodymolecule that binds to CD70, an antibody molecule that binds TIM-3 anddecitabine. In particular embodiments, the combinations described hereincomprise or consist of an antibody molecule that binds to CD70, anantibody molecule that binds to CD47 and azacitidine. In particularembodiments, the combinations described herein comprise or consist of anantibody molecule that binds to CD70, an antibody molecule that bindsCD47 and decitabine. In particular embodiments, the combinationsdescribed herein comprise or consist of an antibody molecule that bindsto CD70, an antibody molecule that binds to IL1RAP and azacitidine. Inparticular embodiments, the combinations described herein comprise orconsist of an antibody molecule that binds to CD70, an antibody moleculethat binds IL1RAP and decitabine.

Without wishing to be bound by theory, combinations incorporating a CD70antibody molecule, an antibody molecule that binds a LSC target and ahypomethylating agent, for example azacitidine or decitabine, arethought to be particularly effective for the treatment of malignancy,particularly myeloid malignancy, due to the combined actions of theactive agents. As described elsewhere herein, CD70, TIM-3, CD47 andIL1RAP have all been identified as targets upregulated on leukemic stemcells. It has also been found that CD70 expression is upregulated on thesurface of AML blasts and lymphocytes from patients treated with thenucleoside metabolic inhibitor azacitidine (see Richardson & Patel(2014) Nat Rev Rheumatol. 10:72-74; Riether et al. (2015) Science TranslMed. 7:1-12; Zhou et al. (2011) Lupus 20:1365-1371, incorporated hereinby reference). It follows, that azacitidine added to the combinationsdescribed herein, for example as a triple combination strategy, mayserve to upregulate CD70 expression on target LSCs, thereby enhancingthe efficacy of the CD70-LSC target double combination therapy.

In certain embodiments, the combinations described herein comprise aninhibitor of PD-1 (also known as “Programmed cell death protein 1” or“CD279”). Alternatively or in addition, the combinations describedherein may comprise an inhibitor of PD-L1 or PD-L2 (ligands of PD-1).

PD-1 and its ligands, particularly PD-L1, have been relativelywell-characterized as immune checkpoint regulators, and dysregulation ofthe PD-1-PD-L1 signaling pathway in the cancer microenvironment has beenidentified as an important means by which tumors suppress the immuneresponse. The receptor PD-1 is typically expressed on a variety ofimmune cells including monocytes, T cells, B cells, dendritic cells andtumor-infiltrating lymphocytes, and the ligand PD-L1 has been found tobe upregulated on a number of different types of tumor cell (seeOhaegbulam et al. (2015) Trends Mol Med. 21(1):24-33, incorporatedherein by reference). The interaction between PD-L1 on tumor cells andPD-1 on immune cells, particularly T cells, creates an immunosuppressivetumor microenvironment via effects at the level of CD8+ cytotoxic Tcells and also via the generation of Treg cells (see Alsaab et al.(2017) Front Pharmacol. Aug 23(8):561, incorporated herein byreference).

Without wishing to be bound by theory, combinations comprising orconsisting of a CD70 antibody molecule, a TIM-3 antibody molecule and aPD-1 inhibitor or PD-L1 inhibitor are thought to be particularlyeffective for the treatment of malignancy, particularly myeloidmalignancy, due to the combined actions of the active agents. As notedabove, CD70 and TIM-3 are immune checkpoint targets, and therefore thecombining of antibody molecules specifically binding to these targetswith an agent or agents that inhibit a third immune checkpoint targetmay be particularly effective for the treatment of malignancy. It hasalso been shown, in a solid tumor model, that combined targeting ofTIM-3 and PD-1 is a particularly effective therapeutic approach (Sakushiet al. 2010. J Exp Med. 207(10):2187-2194). It follows, that PD-1 and/orPD-L1 inhibitors added to the combinations described herein, for exampleas a triple combination strategy, may further enhance the efficacy ofthe CD7O-TIM3 double combination therapy.

The agent capable of inhibiting PD-1 or PD-L1 may be any suitableanti-cancer agent or inhibitor having specificity for PD-1, PD-L1 or thePD1-PD-L1 signaling axis. Many agents capable of inhibiting the activityof PD-1, PD-L1 or the PD1-PD-L1 signaling axis have been developed asreported for example, in Alsaab et al. (supra) (incorporated herein byreference), and any of these agents may be incorporated into thecombinations of the present invention. In certain embodiments, the PD-1and/or PD-L1 inhibitor may be an antibody molecule, for example amonoclonal antibody.

The PD-1 inhibitors for inclusion in the combinations described hereinmay be selected from the group including but not limited to: nivolumab;pembrolizumab; pidilizumab, REGN2810; AMP-224; MED10680; and PDR001. ThePD-L1 inhibitors for inclusion in the combinations described herein maybe selected from the group including but not limited to: atezolizumab;and avelumab.

In certain embodiments, the combinations of the invention comprise orconsist of four active agents: (i) a first antibody moleculespecifically binding CD70; (ii) a second antibody molecule specificallybinding TIM-3; (iii) a hypomethylating agent; and (iv) an agent capableof inhibiting either PD-1 or PD-L1. The hypomethylating agent ispreferably azacitidine. It will be understood that each of the fouractive agents may be selected from any of the specific embodimentsdescribed herein for each active agent.

The combinations described herein may further comprise one or moreadditional anti-cancer agents. In certain embodiments, the one or moreadditional anti-cancer agents are inhibitors of additional immunecheckpoint targets.

For embodiments in accordance with the first aspect of the invention,the combinations may additionally comprise an agent that inhibits SIRPαsignaling. Agents capable of inhibiting SIRPα signaling are describedabove in the context of combinations of the second aspect of theinvention. Any of these agents may be included as an additionalcomponent of the combinations described in accordance with the firstaspect of the invention. For embodiments wherein the agent that inhibitsSIRPα signaling is a SIRPα antibody molecule fusion protein, theantibody molecule to which the SIRPα protein or domain thereof is linkedis preferably an antibody molecule of the combination i.e. an antibodymolecule that binds CD70 or an antibody molecule that binds a LSCtarget. In certain embodiments, the agent that inhibits SIRPα signalingis the immunoglobulin V-like domain of the SIRPα protein and at leastone copy of this domain is fused to the CD70 antibody molecule of thecombination.

In certain embodiments, the combinations of both the first and secondaspects of the invention comprise one or more anti-cancer agents for usein the treatment of myeloid malignancies, for example one or more agentssuitable for use in treating AML. Agents that may be incorporated intothe combinations described herein include but are not limited to:Venetoclax; Vyxeos; Idhifa (or Enasidenib—an isocitrate dehydrogenase(IDH) inhibitor); and Rydapt (midostaurin—a FLT3 inhibitor). In certainembodiments, the combinations additionally comprise Venetoclax. Incertain embodiments, the combinations additionally comprise Vyxeos.

Any of the combinations described herein can be packaged as a kit andoptionally include instructions for use.

E. Methods of Treatment

The combination therapies as described herein are for use in methods oftreating a malignancy in a human subject.

The present invention provides an antibody molecule that binds to CD70for use in the treatment of a malignancy in a human subject, wherein theantibody molecule is administered in combination with a second antibodymolecule that binds to a leukemic stem cell target. The presentinvention also provides an antibody molecule that binds to a leukemicstem cell target for use in the treatment of a malignancy in a humansubject, wherein the antibody molecule is administered in combinationwith a second antibody molecule that binds to CD70. The invention alsoprovides an antibody molecule that binds to CD70 for use in thetreatment of malignancy in a human subject, wherein the antibodymolecule is administered in combination with an agent that inhibitsSIRPα signaling. The invention further provides an agent that inhibitsSIRPα signaling for use in the treatment of malignancy in a humansubject, wherein the agent is administered in combination with anantibody molecule that binds CD70.

The present invention further provides combinations in accordance withthe first and second aspects of the invention for use in the treatmentof a malignancy in a human subject. In a yet further aspect, the presentinvention provides a method for treating a malignancy in a humansubject, said method comprising administering to the subject acombination in accordance with the first or second aspect of theinvention. All embodiments described above in relation to thecombinations of the first and second aspects of the invention areequally applicable to the methods described herein.

The term “malignancy” encompasses diseases in which abnormal cellsproliferate in an uncontrolled manner and invade the surroundingtissues. Malignant cells that have entered the body's blood and lymphsystems are capable of travelling to distal sites in the body andseeding at secondary locations.

In certain embodiment, the methods described herein are for treatingmalignancies comprising the production of cancer progenitor or stemcells expressing CD70, CD27, or both. As noted elsewhere herein,upregulated CD70 expression has been detected in different types ofcancers including renal cell carcinomas, metastatic breast cancers,brain tumors, leukemias, lymphomas and nasopharyngeal carcinomas.Co-expression of CD70 and CD27 has also been detected in malignancies ofthe hematopoietic lineage including acute lymphoblastic lymphoma and Tcell lymphoma. In certain embodiments, the methods described herein arefor the treatment of any of the aforementioned malignancies associatedwith CD70 expression, CD27 expression or both.

In certain embodiment, the methods described herein are for treatingmalignancies comprising the production of cancer progenitor or stemcells expressing one or more the LSC targets to which an antibodymolecule of the combination binds. For example, combinations comprisingan antibody molecule that binds TIM-3 may be used to treatTIM-3-expressing malignancies. Combinations comprising an antibodymolecule that binds CD47 may be used to treat CD47-expressingmalignancies. Combinations comprising an antibody molecule that bindsIL1RAP may be used to treat IL1RAP-expressing malignancies.

In particular embodiments, the methods described herein are for treatingmyeloid malignancies, wherein a myeloid malignancy refers to any clonaldisease of hematopoietic stem or progenitor cells. The myeloidmalignancy treated in accordance with the methods of the invention maybe a newly diagnosed myeloid malignancy or a relapsed/refractory myeloidmalignancy.

As described elsewhere herein, the combinations of the present inventionare thought to be particularly effective for the treatment of myeloidmalignancies, for the reason that CD70, TIM-3, the CD47-SIRPα axis andIL1 RAP have all been identified as key therapeutic targets in myeloidmalignancies, particularly acute myeloid leukemia, see Kikushige et al.(2015) (supra), Riether et al. (2017) (supra), Theocharides et al.(2012) (supra), Ponce et al. (2017) (supra), Agerstam et al. (2015)(supra).

In certain embodiments, the myeloid malignancy is selected from: acutemyeloid leukemia (AML); myelodysplastic syndromes (MDS);myeloproliferative neoplasms (MPN); chronic myeloid leukemia (CML); andchronic myelomonocytic leukemias (CMML). In preferred embodiments, themyeloid malignancy is acute myeloid leukemia (AML).

Myeloid malignancies can be categorized and diagnosed according to theWorld Health Organization (WHO) 2008 classification, taken incombination with the 2016 update to this classification, see inparticular Arber et al. (2016) Blood 127(20):2391-2405, incorporatedherein by reference.

Acute myeloid leukemia (AML) refers to hematopoietic neoplasms involvingmyeloid cells. AML is characterized by clonal proliferation of myeloidprecursors with reduced differentiation capacity. AML patients exhibitan accumulation of blast cells in the bone marrow. Blast cells alsoaccumulate in the peripheral blood of AML patients. Typically AML isdiagnosed if the patient exhibits 20% or more blast cells in the bonemarrow or peripheral blood.

According to the WHO classification, AML in general encompasses thefollowing subtypes:

AML with recurrent genetic abnormalities; AML withmyelodysplasia-related changes; therapy-related myeloid neoplasms;myeloid sarcoma; myeloid proliferations related to Down syndrome;blastic plasmacytoid dendritic cell neoplasm; and AML not otherwisecategorized (e.g. acute megakaryoblastic leukemia, acute basophilicleukemia).

AML can also be categorized according to the French-American-British(FAB) classification, encompassing the subtypes: M0 (acute myeloblasticleukemia, minimally differentiated); M1 (acute myeloblastic leukemia,without maturation); M2 (acute myeloblastic leukemia, with granulocyticmaturation); M3 (promyelocytic, or acute promyelocytic leukemia (APL));M4 (acute myelomonocytic leukemia); M4eo (myelomonocytic together withbone marrow eosinophilia); M5 (acute monoblastic leukemia (M5a) or acutemonocytic leukemia (M5b)); M6 (acute erythroid leukemias, includingerythroleukemia (M6a) and very rare pure erythroid leukemia (M6b)); orM7 (acute megakaryoblastic leukemia).

As used herein, “AML” refers to any of the conditions encompassed by theWHO and/or FAB classifications, unless specified otherwise. Certain AMLsubtypes are considered to be of more favorable prognosis, some ofintermediate prognosis and some of poor prognosis. The skilled person isaware of which subtypes would fall into which risk category.

Myelodysplastic syndrome (MDS) is characterized by dysplasia, cytopeniaand/or abnormal changes in bone marrow cellularity and/or myeloiddifferentiation, for example increased blast cell infiltration.According to the WHO classification, MDS in general encompasses thefollowing subtypes: MDS with single lineage dysplasia (previously called“refractory cytopenia with unilineage dysplasia”, which includesrefractory anemia, refractory neutropenia, and refractorythrombocytopenia); MDS with ring sideroblasts, which includes subgroupswith single lineage dysplasia and multilineage dysplasia (previouslycalled “refractory anemia with ring sideroblasts”); MDS withmultilineage dysplasia (previously called “refractory cytopenia withmultilineage dysplasia”); MDS with excess blasts (MDS-EB, previouslycalled “refractory anemia with excess blasts”), which can be furthersubclassified into MDS-EB-1 and MDS-EB-2 based on blast percentages; MDSwith isolated del(5q); and MDS, unclassified.

MDS can also be categorized according to the French-American-British(FAB) classification, encompassing the subtypes: M9980/3 (refractoryanemia (RA)); M9982/3 (refractory anemia with ring sideroblasts (RARS));M9983/3 (refractory anemia with excess blasts (RAEB)); M9984/3(refractory anemia with excess blasts in transformation (RAEB-T)); andM9945/3 (chronic myelomonocytic leukemia (CMML)).

As used herein, “MDS” refers to any of the conditions encompassed by theWHO and/or FAB classifications, unless specified otherwise. For both AMLand MDS, the WHO categorization is preferred herein.

Myeloproliferative neoplasms (MPN) are similar to MDS but according tothe WHO classification, MPN in general encompasses the followingsubtypes: chronic myeloid leukemia (CML); chronic neutrophilic leukemia(CNL); polycythemia vera (PV); primary myelofibrosis (PMF); essentialthrombocythemia (ET); chronic eosinophilic leukemia, not otherwisespecified; and MPN unclassifiable.

Chronic myelomonocytic leukemia (CMML) and atypical chronic myeloidleukemia (aCML) fall within the category of MDS/MPN disorders accordingto the WHO classification, for the reason that they represent myeloidneoplasms with clinical, laboratory and morphologic features thatoverlap between MDS and MPN.

In certain embodiments, the methods described herein involve monitoringthe patient's blast count i.e. the number of blast cells. As usedherein, “blast cells” or “blasts” refer to myeloblasts or myeloid blastswhich are the myeloid progenitor cells within the bone marrow. Inhealthy individuals, blasts are not found in the peripheral bloodcirculation and there should be less than 5% blast cells in the bonemarrow. In subjects with myeloid malignancies, particularly AML and MDS,there is increased production of abnormal blasts with disrupteddifferentiation potential, and the overproduction of these abnormalblasts can be detected by monitoring the patient's blast count in theperipheral blood circulation or the bone marrow or both.

The proportion of blast cells in the bone marrow or peripheral blood canbe assessed by methods known in the art, for example flow cytometric orcell morphologic assessment of cells obtained from a bone marrow biopsyof the subject, or a peripheral blood smear. The proportion of blasts isdetermined versus total cells in the sample. For example, flow cytometrycan be used to determine the proportion of blast cells using the numberof CD45^(dim), SSC^(low) cells relative to total cell number. By way offurther example, cell morphological assessment can be used to determinethe number of morphologically identified blasts relative to the totalnumber of cells in the field of view being examined.

In certain embodiments are provided methods for reducing the proportionof blasts cells in the bone marrow to less than 25%, less than 20%, forexample less than 10%. In certain embodiments are provided methods forreducing the proportion of blasts cells in the bone marrow to less than5%. In certain embodiments are provided methods for reducing theproportion of blast cells in the bone marrow to between about 5% andabout 25%, wherein the bone marrow blast cell percentage is also reducedby more than 50% as compared with the bone marrow blast cell percentageprior to performing the method (or pretreatment).

In certain embodiments are provided methods for reducing the proportionof blasts cells in the peripheral blood to less than 25%, less than 20%,for example less than 10%. In certain embodiments are provided methodsfor reducing the proportion of blasts cells in the peripheral blood toless than 5%. In certain embodiments are provided methods for reducingthe proportion of blast cells in the peripheral blood to between about5% and about 25%, wherein the peripheral blood blast cell percentage isalso reduced by more than 50% as compared with the peripheral blast cellpercentage prior to performing the method (or pretreatment).

For clinical determination of blast cell percentage, typically cellmorphological (also known as cytomorphology) assessment is preferred.

In particular embodiments, the methods described herein induce acomplete response. In the context of AML treatment, a complete responseor “complete remission” is defined as: bone marrow blasts <5%; absenceof circulating blasts and blasts with Auer rods; absence ofextramedullary disease; ANC>1.0×10⁹/L (1000/μL); platelet count≥100×10⁹/L (100,000/μL), see Döhner et al. (2017) Blood 129(4): 424-447.

The methods may achieve a complete response with platelet recovery i.e.a response wherein the platelet count is >100×10⁹/L (100,000/μL). Themethods may achieve a complete response with neutrophil recovery i.e. aresponse wherein the neutrophil count is >1.0×10⁹/L (1000/μL).Alternatively or in addition, the methods may induce a transfusionindependence of red blood cells or platelets, or both, for 8 weeks orlonger, 10 weeks or longer, 12 weeks or longer.

In particular embodiments, the methods described herein induce a minimalresidual disease (or MRD) status that is negative.

In certain embodiments, the methods described herein induce a completeresponse without minimal residual disease (CR_(MRD).), see Döhner et al.ibid.

The method may achieve a partial response or induce partial remission.In the context of AML treatment, a partial response or partial remissionincludes a decrease of the bone marrow blast percentage of 5% to 25% anda decrease of pretreatment bone marrow blast percentage by at least 50%,see Döhner et al. ibid.

The methods described herein may increase survival. The term “survival”as used herein may refer to overall survival, 1-year survival, 2-yearsurvival, 5-year survival, event-free survival, progression-freesurvival. The methods described herein may increase survival as comparedwith the gold-standard treatment for the particular disease or conditionto be treated. The gold-standard treatment may also be identified as thebest practice, the standard of care, the standard medical care orstandard therapy. For any given disease, there may be one or moregold-standard treatments depending on differing clinical practice, forexample in different countries. The treatments already available formyeloid malignancies are varied and include chemotherapy, radiationtherapy, stem cell transplant and certain targeted therapies.Furthermore, clinical guidelines in both the US and Europe govern thestandard treatment of myeloid malignancies, for example AML; seeO'Donnell et al. (2017) Journal of the National Comprehensive CancerNetwork 15(7):926-957 and Dohner et al. (2017) Blood 129(4):424-447,both incorporated herein by reference.

The methods of the present invention may increase or improve survivalrelative to patients undergoing any of the standard treatments formyeloid malignancy.

The patients or subjects treated in accordance with the methodsdescribed herein, particularly those having AML, may have newlydiagnosed disease, relapsed disease or primary refractory disease. Astandard approach to treatment for newly diagnosed AML patients is the“standard 7+3 intensive chemotherapy” approach characterized by 7 daysof high dose cytarabine followed by 3 days of anthracyclineadministration (e.g. daunorubicin or idarubicin). Intensive chemotherapyis given with the aim of inducing complete remission of AML, typicallywith the intention of the patient undergoing a stem cell transplantfollowing successful chemotherapy.

Standard intensive chemotherapy is associated with significant toxicityand side-effects, meaning it is not suitable for patients unable totolerate these effects. These patients are termed “ineligible forstandard intensive chemotherapy”. A patient may be ineligible forstandard intensive chemotherapy because, for example, they exhibit oneor more comorbidities indicating they would not tolerate the toxicity,or the prognostic factors characterizing their disease indicate anunfavorable outcome of standard intensive chemotherapy. Determination ofan individual patient's eligibility for standard intensive chemotherapywould be performed by a clinician taking into account the individualpatient's medical history and clinical guidelines (e.g. the NationalComprehensive Cancer Network (NCCN) guidelines, incorporated herein byreference). AML patients over the age of 60 are often assessed asineligible for standard intensive chemotherapy, with other factors to beconsidered including the cytogenetics and/or molecular abnormalities ofthe AML being treated.

A patient ineligible for standard intensive chemotherapy may insteadreceive chemotherapy of reduced intensity, such as low dose cytarabine(LDAC). Patients ineligible for standard intensive chemotherapy and forwhom LDAC is not appropriate can receive best supportive care (BSC),including hydroxyurea (HU) and transfusion support.

Patients or subjects treated in accordance with the methods describedherein may be those classified as “ineligible for standard intensivechemotherapy”. The combinations of the invention comprise targetedtherapies that may be predicted to have fewer side-effects. As such,patients deemed ineligible for standard intensive chemotherapy, for anyof the reasons identified above, may be treated with the combinationsaccording to the present invention.

The methods described herein may include a further step of subjectingthe patient or subject to a bone marrow transplant. The methodsdescribed herein may also be used to prepare a patient or subject havinga myeloid malignancy for a bone marrow transplant. As described above,the methods of the present invention may be carried out so as to reducethe absolute or relative numbers of blast cells in the bone marrow orperipheral blood. In certain embodiments, the methods are carried out soas to reduce the blast cell count in the bone marrow and/or peripheralblood prior to transplant. The methods may be used to reduce the blastcell count to less than 5% to prepare the patient or subject for a bonemarrow transplant.

The methods described herein may include administration of furthertherapeutic agents, for example, further anti-cancer agents. In certainembodiments, the methods comprise the administration of one or moreagents for use in treating myeloid malignancies, for example agentssuitable for use in treating AML. Such agents include but are notlimited to: Venetoclax; Vyxeos; Idhifa (or Enasidenib—an IDH inhibitor);and Rydapt (midostaurin—a FLT3 inhibitor).

Incorporation by Reference

Various patents, published patent applications, and publications arecited in the foregoing description and throughout the followingexamples, each of which is incorporated by reference herein in itsentirety.

EXAMPLES

The invention will be further understood with reference to the followingnon-limiting examples.

Example 1

Antibodies specifically binding TIM-3 were generated by immunizing llamawith recombinant human TIM-3 Fc chimera (R&D Systems; Human TIM-3Ser22-Arg200; 2365-TM; Lot HKG081212A) at doses of 80 μg (1^(st) and2^(nd) injection) and 40 μg (injections 3-6) and creating Fab librariesfor screening, as described in, for example, WO2010/001251, incorporatedherein by reference.

The CDR, VH and VL sequences of the Fab clones selected from thelibraries are shown in Tables 2, 3 and 4 below.

TABLE 2  VH and VL sequences of Fabs binding to TIM-3 SEQ SEQ ID IDFab clone VH NO: VL NO: 1A11 EVQLVESGGGLVQPGGSLRLSCAASGFTF 9SYELTQSPSVSVALKQTAKITCGGDNIGSKSAQWY 10 SSYAMSWVRQAPGKGPEWVSHINSGGGNQQKPGQAPVLVIYADSRRPSGIPERFSGSNSGNTA TKYADSVKGRFTISRDNAKNTLYLQMNTLKTLTISGAQAEDEADYYCQVWDSSAAVFGGGTHLTV PEDTAVYYCAKDVSGGYYGTYALDAWGQ LGTQVVVSS 2A2 EVQVQESGGGLVQPGGSLRLSCAASGFTF 11DIQMTQSPSSVIVSAGEKVTINCKSSQSVLDSSNQK 12 SSYAMSWVRQAPGKGLEWVSDINSGGGSNYLAWYQQRLGQSPRLLIYWASTRESGVPDRFSG TVYTDSVKGRFTISRDNAKNTLYLQMNSLKSGSTTDFTLTISSFQPEDAAVYYCQQGYSVPVTFG PDDTAVYYCATGGSYYSYRLFDYWGQGTQGTKVELKR QVTVSS 2A6 QVQLVESGGGLVQPGGSLRLSCAASGFTF 13NFMLTQPPSLSGSLGQSARLTCTLGSGNSIGAHTIS 14 SNYWMYWVRQAPGKGLEWVSTINTNGAITWYQQKAGSPPRYLLNYYSDSSNHQASGVPSRFSG LYADNVKDRFTVSRDNAKNTLYLQMNSLKSSKDDSTNAGLLLISGLQPEDEADYYCAAGDGSGTV EDTAVYYCAKVKLSGYPHPYYAMDYWGKGFGGGTKLTVL TLVTVSS 2A9 QVQLVESGPGLVKPSQTLSLTCTVSGGSIT 15EIVLTQSPSSVTASVGEKVTINCKSSQSVLSSSNQK 16 TSDDAWSWIRQPAGKGLEWMGVIAYDGSTNYLSWYQQRLGQSPRLLITWASTRESGVPDRFSG RYSPSLQSRTSISRDTSKNQFSLQLSSVTPSGSTTDFTLTISSFQPEDAAVYYCQQGYGAPLTFG EDTAVYYCARTKGVGGTWALDAWGQGTLQGTKVELKR VTVSS 2B6 QVQLVESGGGLVQPGGSLRLSCAASGFAF 17QAVVTQEPSLSVSLGGTVTLICGLRSGSVITSNYP 18 SSYDMSWVRQAPGKGLEWVSTINSGGGSGWFKQTPGQAPRTLIFGASSRHSGVPSRYSGSISG TNYADSMKGRFTISRDNAKNTVYLQMNSLKNKAALTITGAEPEDEADYYCALNKGTYTDVFGGGT PEDTAVYYCAARSPYYTRVPLYDYWGQGT KLTVLQVTVSS 2B9 EVQLQESGPGLVKPSQTLSLTCTVSGASVT 19ATMLTQSPGSLSVVPGESASISCKASQSLTHTDGT 20 TRYNYVVSWIRQPPGKGLEWMGAITYSGSTTALYWLQQKPGQRPQLLIYEVSVRASGVPDRFTGS YYSPSLKSRTSISRDTSKNQFTLQLSSVTPEGSGSDFTLKINGVKAEDAGVYYCAQVAYYPTFGQ DTAVYYCATEGSSSTGVSRYSFGSWGQGT GTKVELKQVTVSS 2B10 QVQLQESGPGLVKPSQTLSLTCTVSGGSIT 21QSALTQPPSVSGTLGKTVTISCAGTSSDIGGYNSVS 22 TNRYLVVTWIRQTPGKGLEVVVGAIAYSGRTVVYQQLPGTAPKLLIYEVNKRASGIPDRFSGSKSGN YYSPSLKSRTSISRDTSKNQFTLQLSSVTPETASLSISGLQSEDEADYYCASYRSANNVVFGGGTK DTGVYYCAHFTGWGGYYWGQGTQVTVSS LTVL2C6 QVQLVESGGGLVQPGGSLRLSCAASGFAF 23 QAVVTQEPSLSVSPGGTVTLTCGLSSGSVTTNNYP24 SSYDMSVVVRQAPGKGPEVVVSTINSGGGS GVVFQQTPGQAPRTLIYSTSSRHSGVPSRFSGSISGTSYADSVKGRFTISRDNAKNTVYLQMNSLK NKAALTITGAQPEDEADYYCALDIGSYTAVFGGGTPEDTAVYYCAARSLYYTRVPMYDYWGQGT HLTVL QVTVSK 2D11EVQLVQPGAELRNPGASVKVSCKASGYTF 25 QAVVTQEPSLSVSPGGTVTLTCGLTSGSVTSSNYP 26TMYYIDVVVRQAPGQGLEWMGRIDPEDGG GVVYRQTPGQAPRPLIYNTNSRHPGVPSRYSGSISETKYAQKFQGRVTFTADTSTSTAYVELSSLR NKATLTITGAEPEDEADYYCALHKGSYTAVFGGGTSEDTAVYYCARIPNGGSYYYTPYDYDYWG HLTVL QGTQVTVSS 2D6QVQLQESGGGLVQPGGSLTLSCAASGFFF 27 HSAVTQPPSVSGSPGKAVTISCVGSSSDVGYGDY 28SSYAMSVVVRQAPGKGLEVVVSSISAGGGT VSVVYQQLPGMAPKWYDVEKRASGIPDRFSGSKSSYYADSVKGRFTISRDSAKNTLVLQMNSLK GNTASLTISGLQSEDEADYYCASYRSDSNFVFGGGPEDTAVYYCAKKRQNFWSEGYDSWGQGT THLAVL QVTVSS 2E2QLQLVESGGGLVQPGGSLRLSCAASGFTF 29 DIVMTQSPSSLSASLGDRVTITCQASQSISSYLAVVY30 GSYDMSVVVRQAPGKGPEVVVSRITSGGGS QQKPGQGPKLLIYGASRLEPGVPSRFSGSGSGTSFTYADSVKGRFTISRDNAKNTLSLQMNSLKS TLTISGVEAEDLATYYCLQDYSWPYSFGSGTRLEIKEDTAVYYCAAGQYSDGYYPYDYWGQGTQ VTVSS 2E7 ELQVVESGGGLVQPGGSLRLSCAASGFTF 31DVVLTQTPGSLSVVPGESASISCKASQSLIHIDGKT 32 GSYDMSWHRQAPRKGPEVVVSTISAGGGRYLYWLLQKPGRRPELLIYQVSNHESGVPDRFTGSG TYYADSVKGRFTISRDNAKNTLYLQMNSLKSGTDFTLKISGVKAEDAGVYYCAQATYYPSFGSGT PEDTAVYFCTKIVLDSWGQGTQVTVSS RLEIK2E9 EVQLVESGGGLVQPGGSLRLSCAASGFTF 33 DIVMTQSPSSVTASVGEKVTINCKSSQSVVSGSNQ34 DDYTMSVVVRQVPGKGLEWISGISGNGGRT KSYLNVVYQQRPGQPPRLLIYYASTQESGIPDRFSGDYVEPIEGRFTISRDNAKNTLYLQMNSLKSE SGSTTDFTLTISSVQPEDAAVYYCQQAYSAPYNFGDTAVYYCAKTSPQSLDYWGQGTQVTVSS SGTRLEIK 2F8 EVQLVESGGGLVQPGGSLRLSCAASGFTF35 DVVLTQTPGSLSVVPGESASISCKASQSLVHTDGK 36 GSYDMSVVVRQAPGKGPEVVVSTISAGGGRTYVYWLLQKPGQRPHLLIYQVSNHESGVPDRFTGS TYYADSVKDRFTISRDNAKNTLYLQMNSLKGSGTDFTLKISGVKAEDAGVYYCAQATYYPSFGSG PEDTAVYYCAKVVIDYWGQGTQVTVSS TRLEIK2G6 EVQLVESGGGLVQPGGSLRLSCAASGFTF 37 QAVLTQPPSVSGSPGQRFTISCTGSNRNIGNNYVN38 SSYSMSVVVRQAPGKGPEVVVSGINTSGGT VVYQQLPGTAPKLLIYSDNLRTSGVPARFSASKSGTTSYAASVKGRFTVSRDNAKNTLSLQMNSLE TSSLTISGLQAEDEAVYYCSSWDDSLSGAVFGGGTPEDTAVYYCVKHIRWSGSNYYYYGMDYW HLTVL GKGTLVTVSS

TABLE 3  Heavy chain CDR sequences of Fabs binding to TIM-3 SEQ SEQ SEQFab  ID ID ID clone CDR1 NO: CDR2 NO: CDR3 NO: 1A11 SYAMS 39HINSGGGNTKYA 40 DVSGGYYGTYA 41 DSVKG LDA 2A2 SYAMS 39 DINSGGGSTVYT 42GGSYYSYRLFDY 43 DSVKG 2A6 NYWMY 44 TINTNGAITLYA 45 VKLSGYPHPYY 46 DNVKDAMDY 2A9 TSDDAWS 47 VIAYDGSTRYSP 48 TKGVGGTWALDA 49 SLQS 2B6 SYDMS 50TINSGGGSTNYA 51 RSPYYTRVPLY 52 DSMKG DY 2B9 TRYNYWS 53 AITYSGSTYYSP 54EGSSSTGVSRY 55 SLKS SFGS 21310 TNRYLWT 56 AIAYSGRTYYSP 57 FTGWGGYY 58SLKS 2C6 SYDMS 50 TINSGGGSTSYA 59 RSLYYTRVPMY 60 DSVKG DY 2D11 MYYID 61RIDPEDGGTKYA 62 IPNGGSYYYTPY 63 QKFQG DYDY 2D6 SYAMS 39 SISAGGGTSYYA 64KRQNFWSEGYDS 65 DSVKG 2E2 SYDMS 50 RITSGGGSTYAD 66 GQYSDGYYPYDY 67 SVKG2E7 SYDMS 50 TISAGGGRTYYA 68 IVLDS 69 DSVKG 2E9 DYTMS 70 GISGNGGRTDYV 71TSPQSLDY 72 EPIEG 2F8 SYDMS 50 TISAGGGRTYYA 73 VVIDY 74 DSVKD 2G6 SYSMS75 GINTSGGTTSYA 76 HIRWSGSNYYY 77 ASVKG YGMDY

TABLE 4  Light chain CDR sequences of Fabs binding to TIM-3 SEQ SEQ SEQFab  ID ID ID clone CDR1 NO: CDR2 NO: CDR3 NO: 1A11 GGDNIGSKSAQ 78ADSRRPS 79 QVWDSSAAV 80 2A2 KSSQSVLDSSNQKNYLA 81 WASTRES 82 QQGYSVPVT 832A6 TLGSGNSIGAHTIS 84 YYSDSSN 85 AAGDGSGTV 86 HQASGV 2A9KSSQSVLSSSNQKNYLS 87 WASTRES 82 QQGYGAPLT 88 2B6 GLRSGSVTTSNYPG 89GASSRHS 90 ALNKGTYTDV 91 2B9 KASQSLTHTDGTTALY 92 EVSVRAS 93 AQVAYYPT 942B10 AGTSSDIGGYNSVS 95 EVNKRAS 96 ASYRSANNVV 97 2C6 GLSSGSVTTNNYPG 98STSSRHS 99 ALDIGSYTAV 100 2D11 GLTSGSVTSSNYPG 101 NTNSRHP 102 ALHKGSYTAV103 2D6 VGSSSDVGYGDYVS 104 DVEKRAS 105 ASYRSDSNFV 106 2E2 QASQSISSYLA107 GASRLEP 108 LQDYSWPYS 109 2E7 KASQSLIHIDGKTYLY 110 QVSNHES 111AQATYYPS 112 2E9 KSSQSVVSGSNQKSYLN 113 YASTQES 114 QQAYSAPYN 115 2F8KASQSLVHTDGKTYVY 116 QVSNHES 111 AQATYYPS 117 2G6 TGSNRNIGNNYVN 118SDNLRTS 119 SSWDDSLSGAV 120

The Fabs shown in the tables above were characterized with respect totheir TIM-3 binding by Biacore analysis and by ELISA. The results areshown in Table 5 below.

TABLE 5 Binding of Fab clones to TIM-3 as measured by Biacore or ELISA.Fab clone Off-rate KD (1/s) EC50 (ng/ml) 2G6 3.72E−05 13.3 2D11 veryhigh n/a 2A6 1.51E−04 10.36 2B9 4.87E−05 8.505 1A11 2.48E−05 10.05 2C61.04E−05 8.212 2B6 3.64E−05 8.518 2D6 1.66E−04 7.169 2E2 very high 121.12A9 high 11.39 2A2 4.41E−05 8.047 2B10 1.85E−05 14.01 2E9 6.64E−05 14.862E7 7.62E−05 13.29 2F8 8.53E−05 18.07

Example 2

Antibodies specifically binding IL1RAP were generated by immunizingllama with recombinant human IL-1RAP/IL-1 R3 Fc Chimera Protein (R&DSystems: Ser21 Glu359/C-terminus HIS-tagged; Cat No. 676-CP) andcreating Fab libraries for screening, as described in, for example,WO2010/001251, incorporated herein by reference.

The CDR, VH and VL sequences of the Fab clones selected from thelibraries are shown in Tables 6, 7 and 8 below.

TABLE 6  VH and VL sequences of Fabs binding to IL1RAP SEQ SEQ Fab  IDID clone VH NO: VL NO: 1F10 QVQLVESGGGLVQPGGSL 121 QAVLTQLPSVSGSPGQK 122RLSCAASGFIFINYGMHW ITISCTGSSSNIGGGYS VRQAPGKGLEWVSAVNSGVQWFQHLPGTPPKLLIY GASTDYADSVKGRFTISR GNSNRASGVPDRFSGSKDDAKNTLYLQMNSLKSED SGSSASLTITGLQAEDE TAVYYCVKGWFYGIHYWGADYYCESYDDWLKGRGF KGTLVTVSS GGGSKLTVL 1C1 QVQLVESGPGLVKPSQTL 123QSVLTQPPSVSGSPGKT 124 SLTCTVSGGSITTNYYSW VTISCAGTSSDVGYGNYIWIRQPPGKGLEWMGASV VSWYQQLPGMAPKWYDV YSGSTFYSPSLKNTSISKDIRASGIADRFSGSKSG DTAQNQFTLQLRSVTPED NTASLTISGLQSEDEADTAVYYCARASSAHWGSSF YYCASYRTNNNAVFGGG ISIDYWGQGTQVTVSS THLTVL

TABLE 7  Heavy chain CDR sequences of Fabs binding to IL1RAP SEQ SEQ SEQFab  ID ID ID clone CDR1 NO: CDR2 NO: CDR3 NO: 1F10 NYGMH 125AVNSGGASTDYADSVKG 126 GWFYGIHY 127 1C1 TNYYSWI 128 ASVYSGSTFYSPSLKN 129ASSAHWGS 130 SFISIDY

TABLE 8  Heavy chain CDR sequences of Fabs binding to IL1RAP SEQ SEQ SEQFab  ID ID ID clone CDR1 NO: CDR2 NO: CDR3 NO: 1F10 TGSSSNIGGGYSVQ 131GNSNRAS 132 ESYDDWLKGRG 133 1C1 AGTSSDVGYGNYVS 134 DVDIRAS 135ASYRTNNNAV 136

Example 3 Combined Efficacy of Anti-TIM-3 and Anti-CD70 AntibodiesMeasured by ADCP Activity

The combined efficacy of anti-TIM-3 and anti-CD70 antibodies wasassessed by measuring antibody-dependent cellular phagocytosis(ADCP)-mediated killing of the AML-derived cell line BDCM. BDCM cellswith PKH26-labelled cell membranes were treated with differentconcentrations of the CD70-targeting antibody ARGX-110 alone or incombination with 10 pg/ml of the anti-TIM-3 antibodies clones 1A10 and2B11 (human IgG1)—see Example 1. Phagocytosis-capable macrophages weredifferentiated from monocytic THP-1 cell line by PMA treatment.Activated macrophages were added to the BDCM cells pre-treated withantibodies and co-incubated with the cancer cells for one hour at 37° C.After washing, macrophages were stained with anti-CD11b-FITC antibodiesand flow cytometry analysis was performed in order to estimate thenumber of macrophages with engulfed cancer cells (PKH26⁺/CD11b⁺ doublepositive macrophages).

As shown in FIG. 1, pre-treatment of CD70 and TIM-3-expressing BDCMcells with ARGX-110 and anti-TIM3 antibodies caused a significantincrease in phagocytosis of cancer cells by macrophages. This increasewas seen in comparison to the treatment of cells with ARGX-110 alone.The combined efficacy of anti-TIM3 and anti-CD70 antibodies inADCP-mediated killing of AML cells was shown in a dose-dependent manner.

Example 4 Combined Efficacy of Anti-IL1RAP and Anti-CD70 AntibodiesMeasured by ADCP Activity

The combined efficacy of anti-IL1RAP and anti-CD70 antibodies wasassessed by measuring antibody-dependent cellular phagocytosis(ADCP)-mediated killing of AML cell lines (MV4-11, U937 and THP-1).PKH126-stained AML cell lines (MV4-11, U937, THP-1) were treated withdifferent concentrations of ARGX-110 alone or in combination with 10μg/ml or 1 μg/ml anti-IL1RAP antibodies (mouse IgG1 clone 89412; IgG2aclone 7E4G1E8 mAbs; and human monoclonal IgG1 antibodies-clones 1C1 and1F10, see Example 2). The assay was performed as described in Example 3above. The results are shown in FIG. 2. Phagocytosis background values,measured in the absence of any treatment, have been subtracted.

As shown in FIG. 2, pre-treatment of CD70- and IL1RAP-expressing AMLcell lines with ARGX-110 and anti-IL1RAP antibodies caused significantincreases in phagocytosis of cancer cells by macrophages. Theseincreases were seen in comparison with conditions where cancer cellswere only treated with ARGX-110. Additive effects of co-treatment wereshown in a dose-dependent manner. Moreover, synergistic efficacy wasobserved when MV4-11 cells were treated with combinations of 1 or 10μg/ml ARGX-110 plus 1C1 or 1F10 antibodies.

Example 5 Combined Efficacy of Anti-CD47 and Anti-CD70 AntibodiesMeasured by ADCP Activity

The combined efficacy of anti-CD47 and anti-CD70 antibodies was assessedin a similar manner to that described above in Examples 3 and 4.PKH126-stained AML cell lines (MV4-11, THP-1, GDM-1, U937 and MC-1010)were treated with different concentrations of ARGX-110 alone or incombination with 10 μg/ml or 1 μg/ml of anti-CD47 antibodies (mouse IgG1clone B6H12 and clone CC2C6, and mouse IgG2b clone BRIC126). The ADCPassay was performed as described above.

As shown in FIG. 3A, pre-treatment of CD70- and CD47- expressing AMLcells with ARGX-110 and anti-CD47 antibodies caused increases inphagocytosis of cancer cells by macrophages in the case of several ofthe AML cell lines. The effect of co-treatment with ARGX-110 andblocking B6H12 antibody (which blocks the interaction between CD47 andSIRPα, thereby promoting phagocytosis) was also shown in adose-dependent manner using MC-1010 cells (FIG. 3B).

Example 6 Combined Efficacy of Anti-TIM-3 and Anti-CD70 AntibodiesMeasured by CDC Activity

The combined efficacy of anti-TIM-3 and anti-CD70 antibodies wasassessed by measuring complement-dependent cytotoxicity (CDC). BDCMcells were treated with different concentrations of ARGX-110 alone or incombination with 10 μg/ml of anti-TIM-3 antibodies (1A11 and 2610clones—see Example 1). Pre-treated cells were incubated with 10% babyrabbit complement (COM) for one hour at room temperature. One volume ofPBS with propidium iodide (PI) was added and samples were incubated inthe dark for fifteen minutes to stain dead cells. Determination of cellnumber and propidium iodide positive cells was performed by flowcytometry (FACS Canto II). The results are shown in FIG. 4.

Co-treatment of BDCM cells with ARGX-110 and anti-TIM3 antibodies causedan increase in complement-dependent cell death. Synergistic effects ofthe combinations of ARGX-110 and anti-TIM-3 antibodies were observed atconcentrations of ARGX-110 between 0.37 and 0.125 μg/ml, whereasARGX-110 alone was able to induce the cell death from 1.11 μg/mlconcentration. These synergistic effects of the co-treatment were shownin a dose-dependent manner. Neither of the anti-TIM-3 antibodies wereable to cause complement-dependent lysis when they were used alone inthe assay.

Example 7 Combined Efficacy of Anti-IL1RAP and Anti-CD70 AntibodiesMeasured by CDC Activity

The combined efficacy of anti-IL1RAP and anti-CD70 antibodies wasassessed by measuring complement-dependent cytotoxicity (CDC). AML celllines (MV4-11 and NOMO-1) were treated with different concentrations ofARGX-110 alone or in combination with 10 pg/ml of anti-IL1RAP antibodies(1C1 and 1F10 clones) and CDC assay was performed as described inExample 6. The results are shown in FIG. 5.

Co-treatment with ARGX-110 and anti-IL1RAP antibodies increased thecomplement-dependent cell death (FIG. 5, dark bars) of both cell lines.The MV4-11 cell line was resistant to the treatment with ARGX-110 andanti-IL1RAP alone. However, a synergistic effect was observed with theco-treatment, causing lysis of MV4-11 cells in a dose-dependent manner.ARGX-110-sensitive cell line NOMO-1 showed a dose-dependent effect afterco-treatment with ARGX-110 and anti-IL1RAP antibodies in comparison withtreatment with ARGX-110 alone. In the case of NOMO-1 cells, monotherapywith anti-IL1RAP antibodies induced a limited complement-dependentcytotoxicity when the antibodies were used at 10 μg/ml concentration.

Example 8 Combined Efficacy of Anti-CD47 and Anti-CD70 AntibodiesMeasured by CDC Activity

The combined efficacy of anti-CD47 and anti-CD70 antibodies was assessedby measuring complement-dependent cytotoxicity (CDC). MV4-11 and NOMO-1cell lines were treated with different concentrations of ARGX-110 andCDC-capable anti-CD47 antibody BRIC126 (mouse IgG2b) alone or incombinations. The CDC assay was performed as described above. Theresults are shown in FIG. 6.

Co-treatment of the AML cell lines with ARGX-110 and BRIC126 increasedthe complement-dependent cell death in both cell lines, whereas ananti-CD47 blocking mouse IgG1 antibody was not able to induce complementresponse (B6H12 clone) (data not shown). The effect of co-treatment withARGX-110 and BRIC126 was observed in a dose-dependent manner with anoptimal concentration of BRIC126 between 0.041 and 0.123 μg/ml. TheMV4-11 cell line is only weakly responsive to ARGX-110 and therefore ashigh as 10 μg/ml concentration was needed to obtain a combined effect.In the ARGX-110-sensitive cell line, NOMO-1, the cells were lysed bycomplement at a ten times lower concentration of ARGX-110 alone.Furthermore, adding BRIC126, at about 0.1 μg/ml, further augmented celllysis by complement. Monotherapy with BRIC126 at higher concentrationswas also able to induce complement-dependent cytotoxicity.

Example 9 Combined Efficacy of Anti-TIM-3, Anti-IL1RAP or Anti-CD47Antibodies and Anti-CD70 Antibodies Measured by ADCC Activity

The efficacy of the anti-CD70 antibody ARGX-110 in combination witheither anti-TIM-3 antibodies, anti-IL1RAP antibodies or anti-CD47antibodies was measured by antibody-dependent cellular cytotoxicity(ADCC). The ADCC activity of the following antibody combinations wasinvestigated:

-   -   1. ARGX-110 (anti-CD70) and 2610 (anti-TIM-3)    -   2. ARGX-110 (anti-CD70) and 1F10 (anti-IL1RAP)    -   3. ARGX-110 (anti-CD70) and CC2C6 (anti-CD47)

For all combinations tested, ADCC was measured according to thefollowing protocol. Healthy peripheral blood mononuclear cells (PBMCs)were treated with recombinant IL-2 (200 IU/mL) for 15 hours. Cell linesBDCM and NOMO-1 were used as CD70-positive target cells, also expressingCD47 and TIM-3 or IL1RAP1 respectively. Target cells (3E4 cells) wereco-cultured with PBMCs (3E5cells) in the presence of antibodies in RPMI1640 medium with 10% FCS (96-well plate). The effector/target (E/T)ratio was 1/1. A dilution series of ARGX-110 (0-10 μg/mL) alone or incombination with the antibodies 2B10 (anti-TIM-3), 1F10 (anti-IL1RAP) orCC2C6 (anti-CD47) at a concentration of 10 and 1 μg/mL were applied. Allantibodies except CC2C6 (mouse IgG1) were human IgG1 isotype. After 48hours of incubation, the cells were analyzed by flow cytometry and the %lysis was measured based on the number of target cells (CD33⁺ CD3⁻CD16⁻) remaining. The results are shown in FIGS. 7 (anti-CD70 +anti-TIM-3), 8 (anti-CD70 + anti-IL1RAP) and 9 (anti-CD70+anti-CD47).

As shown in FIG. 7, both anti-CD70 and anti-TIM-3 antibodies showedstrong ADCC activity alone and reached a maximum cell lysis of 50-70% ata concentration of 1 μg/mL or higher. A combined activity was observedat lower concentration of ARGX-110 (<0.1 μg/ml).

As shown in FIG. 8, a combined ADCC activity was achieved across theARGX-110 dose range when combined with the anti-IL1RAP antibody 1F10 at1 μg/ml. This combination reached a maximum cell lysis of 70% at thehighest ARGX-110 concentration tested (10 μg/ml). The anti-IL1RAPantibody 1F10 showed a strong ADCC activity alone at 10 μg/ml whichresulted in 60-70% cell lysis.

As shown in FIG. 9, combined ADCC activity was achieved across theARGX-110 dose range when combined with anti-CD47 antibody CC2C6 at 1 or10 μg/ml. This combination reached a maximum cell lysis of 80% at 0.1μg/mL or higher concentrations of ARGX-110.

1. A combination comprising an antibody molecule that binds to CD70 andat least one antibody molecule that binds to a leukemic stem cell (LSC)target.
 2. The combination of claim 1, wherein the leukemic stem celltarget is selected from the group consisting of: TIM-3; Galectin-9;CD47; IL1RAP; LILRB2; CLL-1; CD123; CD33; SAIL; GPR56; CD44; E-selectin;CXCR4; CD25; CD32; PR1; WT1; ADGRE2; CCR1; TNFRSF1B and CD96.
 3. Thecombination of claim 1, wherein the antibody molecule that binds to CD70comprises a variable heavy chain domain (VH) and a variable light chaindomain (VL) wherein the VH and VL domains comprise the CDR sequences:HCDR3 comprising or consisting of SEQ ID NO: 3; HCDR2 comprising orconsisting of SEQ ID NO: 2; HCDR1 comprising or consisting of SEQ ID NO:1; LCDR3 comprising or consisting of SEQ ID NO: 7; LCDR2 comprising orconsisting of SEQ ID NO: 6; and LCDR1 comprising or consisting of SEQ IDNO:
 5. 4. The combination of claim 3, wherein the antibody molecule thatbinds to CD70 comprises a VH domain comprising an amino acid sequence atleast 70% identical to SEQ ID NO: 4 and a VL domain comprising an aminoacid sequence at least 70% identical to SEQ ID NO:
 8. 5. The combinationof claim 4, wherein the antibody molecule that binds to CD70 isARGX-110.
 6. (canceled)
 7. The combination of claim 1, wherein theleukemic stem cell target is TIM-3. 8.-10. (canceled)
 11. Thecombination of claim 1, wherein the combination comprises an antibodymolecule that binds to a first leukemic stem cell target and an antibodymolecule that binds to a second leukemic stem cell target, wherein thefirst and second leukemic stem cell targets are different. 12.(canceled)
 13. The combination of claim 11, wherein the first leukemicstem cell target is selected from the group consisting of: TIM-3,Galectin-9, CD47, IL1RAP, and LILRB2.
 14. The combination of claim 11,wherein the second leukemic stem cell target is selected from the groupconsisting of: TIM-3; Galectin-9; CD47; IL1RAP and LILRB2.
 15. Thecombination of claim 11, wherein the first leukemic stem cell target isTIM-3 and the second leukemic stem cell target is CD47.
 16. Thecombination of claim 11, wherein the first leukemic stem cell target isTIM-3 and the second leukemic stem cell target is IL1RAP.
 17. Thecombination of claim 1 comprising an antibody molecule that bindsIL1RAP, wherein said antibody molecule results in reduced NF-κBsignaling; reduced Wnt signaling/β-catenin signaling; reduced sternnessof AML, cells; or a combination thereof.
 18. The combination of claim 11comprising an antibody molecule that binds TIM-3, wherein said antibodymolecule results in reduced NF-κB signaling; reduced Wntsignaling/β-catenin signaling; reduced sternness of AML cells; or acombination thereof.
 19. The combination of claim 1, comprising anantibody molecule that binds TIM-3, wherein said antibody molecule thatbinds TIM-3 inhibits the interaction of TIM-3 with one or more TIM-3interacting proteins.
 20. The combination of claim 19, wherein the TIM-3interacting proteins are selected from the group consisting of:CEACAM-1, HMGB-1, phosphatidylserine, Galectin-9, LILRB2, and anycombination thereof. 21.-25. (canceled)
 26. The combination of claim 1,comprising an antibody molecule that binds TIM-3, wherein said antibodymolecule is selected from the group consisting of antibody moleculescomprising a combination of variable heavy chain CDR3 (HCDR3), variableheavy chain CDR2 (HCDR2) and variable heavy chain CDR1 (HCDR1), variablelight chain CDR3 (LCDR3), variable light chain CDR2 (LCDR2) and variablelight chain CDR1 (LCDR1) selected from the following: (i) HCDR3comprising SEQ ID NO: 41; HCDR2 comprising SEQ ID NO: 40; HCDR1comprising SEQ ID NO: 39; LCDR3 comprising SEQ ID NO: 80; LCDR2comprising SEQ ID NO: 79; and LCDR1 comprising SEQ ID NO: 78; (ii) HCDR3comprising SEQ ID NO: 43; HCDR2 comprising SEQ ID NO: 42; HCDR1comprising SEQ ID NO: 39; LCDR3 comprising SEQ ID NO: 83; LCDR2comprising SEQ ID NO: 82; and LCDR1 comprising SEQ ID NO: 81; (iii)HCDR3 comprising SEQ ID NO: 46; HCDR2 comprising SEQ ID NO: 45; HCDR1comprising SEQ ID NO: 44; LCDR3 comprising SEQ ID NO: 86; LCDR2comprising SEQ ID NO: 85; and LCDR1 comprising SEQ ID NO: 84; (iv) HCDR3comprising SEQ ID NO: 49; HCDR2 comprising SEQ ID NO: 48; HCDR1comprising SEQ ID NO: 47; LCDR3 comprising SEQ ID NO: 88; LCDR2comprising SEQ ID NO: 82; and LCDR1 comprising SEQ ID NO: 87; (v) HCDR3comprising SEQ ID NO: 52; HCDR2 comprising SEQ ID NO: 51; HCDR1comprising SEQ ID NO: 50; LCDR3 comprising SEQ ID NO: 91; LCDR2comprising SEQ ID NO: 90; and LCDR1 comprising SEQ ID NO: 89; (vi) HCDR3comprising SEQ ID NO: 55; HCDR2 comprising SEQ ID NO: 54; HCDR1comprising SEQ ID NO: 53; LCDR3 comprising SEQ ID NO: 94; LCDR2comprising SEQ ID NO: 93; and LCDR1 comprising SEQ ID NO: 92; (vii)HCDR3 comprising SEQ ID NO: 58; HCDR2 comprising SEQ ID NO: 57; HCDR1comprising SEQ ID NO: 56; LCDR3 comprising SEQ ID NO: 97; LCDR2comprising SEQ ID NO: 96; and LCDR1 comprising SEQ ID NO: 95; (viii)HCDR3 comprising SEQ ID NO: 60; HCDR2 comprising SEQ ID NO: 59; HCDR1comprising SEQ ID NO: 50; LCDR3 comprising SEQ ID NO: 100; LCDR2comprising SEQ ID NO: 99; and LCDR1 comprising SEQ ID NO: 98; (ix) HCDR3comprising SEQ ID NO: 63; HCDR2 comprising SEQ ID NO: 62; HCDR1comprising SEQ ID NO: 61; LCDR3 comprising SEQ ID NO: 103; LCDR2comprising SEQ ID NO: 102; and LCDR1 comprising SEQ ID NO: 101; (x)HCDR3 comprising SEQ ID NO: 65; HCDR2 comprising SEQ ID NO: 64; HCDR1comprising SEQ ID NO: 39; LCDR3 comprising SEQ ID NO: 106; LCDR2comprising SEQ ID NO: 105; and LCDR1 comprising SEQ ID NO: 104; (xi)HCDR3 comprising SEQ ID NO: 67; HCDR2 comprising SEQ ID NO: 66; HCDR1comprising SEQ ID NO: 50; LCDR3 comprising SEQ ID NO: 109; LCDR2comprising SEQ ID NO: 108; and LCDR1 comprising SEQ ID NO: 107; (xii)HCDR3 comprising SEQ ID NO: 69; HCDR2 comprising SEQ ID NO: 68; HCDR1comprising SEQ ID NO: 50; LCDR3 comprising SEQ ID NO: 112; LCDR2comprising SEQ ID NO: 111; and LCDR1 comprising SEQ ID NO: 110; (xiii)HCDR3 comprising SEQ ID NO: 72; HCDR2 comprising SEQ ID NO: 71; HCDR1comprising SEQ ID NO: 70; LCDR3 comprising SEQ ID NO: 115; LCDR2comprising SEQ ID NO: 114; and LCDR1 comprising SEQ ID NO: 113; (xiv)HCDR3 comprising SEQ ID NO: 74; HCDR2 comprising SEQ ID NO: 73; HCDR1comprising SEQ ID NO: 50; LCDR3 comprising SEQ ID NO: 117; LCDR2comprising SEQ ID NO: 111; and LCDR1 comprising SEQ ID NO: 116; and (xv)HCDR3 comprising SEQ ID NO: 77; HCDR2 comprising SEQ ID NO: 76; HCDR1comprising SEQ ID NO: 75; LCDR3 comprising SEQ ID NO: 120; LCDR2comprising SEQ ID NO: 119; and LCDR1 comprising SEQ ID NO:
 118. 27. Thecombination of claim 1, comprising an antibody molecule that bindsTIM-3, wherein said antibody molecule is selected from an antibodymolecule comprising or consisting of a variable heavy chain domain (VH)and a variable light chain domain (VL) selected from the following: a VHdomain comprising or consisting of the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99%identity thereto, and a VL domain comprising or consisting of the aminoacid sequence of SEQ ID NO: 10 or an amino acid sequence having at least80%, 90%, 95%, 98% 99% identity thereto; (ii) a VH domain comprising orconsisting of the amino acid sequence of SEQ ID NO: 11 or an amino acidsequence having at least 80%, 90%, 95%, 98% 99% identity thereto, and aVL domain comprising or consisting of the amino acid sequence of SEQ IDNO: 12 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99%identity thereto; (iii) a VH domain comprising or consisting of theamino acid sequence of SEQ ID NO: 13 or an amino acid sequence having atleast 80%, 90%, 95%, 98% 99% identity thereto, and a VL domaincomprising or consisting of the amino acid sequence of SEQ ID NO: 14 oran amino acid sequence having at least 80%, 90%, 95%, 98% 99% identitythereto; (iv) a VH domain comprising or consisting of the amino acidsequence of SEQ ID NO: 15 or an amino acid sequence having at least 80%,90%, 95%, 98% 99% identity thereto, and a VL domain comprising orconsisting of the amino acid sequence of SEQ ID NO: 16 or an amino acidsequence having at least 80%, 90%, 95%, 98% 99% identity thereto; (v) aVH domain comprising or consisting of the amino acid sequence of SEQ IDNO: 17 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99%identity thereto, and a VL domain comprising or consisting of the aminoacid sequence of SEQ ID NO: 18 or an amino acid sequence having at least80%, 90%, 95%, 98% 99% identity thereto; (vi) a VH domain comprising orconsisting of the amino acid sequence of SEQ ID NO: 19 or an amino acidsequence having at least 80%, 90%, 95%, 98% 99% identity thereto, and aVL domain comprising or consisting of the amino acid sequence of SEQ IDNO: 20 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99%identity thereto; (vii) a VH domain comprising or consisting of theamino acid sequence of SEQ ID NO: 21 or an amino acid sequence having atleast 80%, 90%, 95%, 98% 99% identity thereto, and a VL domaincomprising or consisting of the amino acid sequence of SEQ ID NO: 22 oran amino acid sequence having at least 80%, 90%, 95%, 98% 99% identitythereto; (viii) a VH domain comprising or consisting of the amino acidsequence of SEQ ID NO: 23 or an amino acid sequence having at least 80%,90%, 95%, 98% 99% identity thereto, and a VL domain comprising orconsisting of the amino acid sequence of SEQ ID NO: 24 or an amino acidsequence having at least 80%, 90%, 95%, 98% 99% identity thereto; (ix) aVH domain comprising or consisting of the amino acid sequence of SEQ IDNO: 25 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99%identity thereto, and a VL domain comprising or consisting of the aminoacid sequence of SEQ ID NO: 26 or an amino acid sequence having at least80%, 90%, 95%, 98% 99% identity thereto; (x) a VH domain comprising orconsisting of the amino acid sequence of SEQ ID NO: 27 or an amino acidsequence having at least 80%, 90%, 95%, 98% 99% identity thereto, and aVL domain comprising or consisting of the amino acid sequence of SEQ IDNO: 28 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99%identity thereto; (xi) a VH domain comprising or consisting of the aminoacid sequence of SEQ ID NO: 29 or an amino acid sequence having at least80%, 90%, 95%, 98% 99% identity thereto, and a VL domain comprising orconsisting of the amino acid sequence of SEQ ID NO: 30 or an amino acidsequence having at least 80%, 90%, 95%, 98% 99% identity thereto; (xii)a VH domain comprising or consisting of the amino acid sequence of SEQID NO: 31 or an amino acid sequence having at least 80%, 90%, 95%, 98%99% identity thereto, and a VL domain comprising or consisting of theamino acid sequence of SEQ ID NO: 32 or an amino acid sequence having atleast 80%, 90%, 95%, 98% 99% identity thereto; (xiii) a VH domaincomprising or consisting of the amino acid sequence of SEQ ID NO: 33 oran amino acid sequence having at least 80%, 90%, 95%, 98% 99% identitythereto, and a VL domain comprising or consisting of the amino acidsequence of SEQ ID NO: 34 or an amino acid sequence having at least 80%,90%, 95%, 98% 99% identity thereto; (xiv) a VH domain comprising orconsisting of the amino acid sequence of SEQ ID NO: 35 or an amino acidsequence having at least 80%, 90%, 95%, 98% 99% identity thereto, and aVL domain comprising or consisting of the amino acid sequence of SEQ IDNO: 36 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99%identity thereto; and (xv) a VH domain comprising or consisting of theamino acid sequence of SEQ ID NO: 37 or an amino acid sequence having atleast 80%, 90%, 95%, 98% 99% identity thereto, and a VL domaincomprising or consisting of the amino acid sequence of SEQ ID NO: 38 oran amino acid sequence having at least 80%, 90%, 95%, 98% 99% identitythereto.
 28. The combination of claim 1, comprising an antibody moleculethat binds IL1RAP, wherein said antibody molecule is selected from thegroup consisting of antibody molecules comprising a combination ofvariable heavy chain CDR3 (HCDR3), variable heavy chain CDR2 (HCDR2) andvariable heavy chain CDR1 (HCDR1), variable light chain CDR3 (LCDR3),variable light chain CDR2 (LCDR2) and variable light chain CDR1 (LCDR1)selected from the following: (i) HCDR3 comprising SEQ ID NO: 127; HCDR2comprising SEQ ID NO: 126; HCDR1 comprising SEQ ID NO: 125; LCDR3comprising SEQ ID NO: 133; LCDR2 comprising SEQ ID NO: 132; and LCDR1comprising SEQ ID NO: 131; and (ii) HCDR3 comprising SEQ ID NO: 130;HCDR2 comprising SEQ ID NO: 129; HCDR1 comprising SEQ ID NO: 128; LCDR3comprising SEQ ID NO: 136; LCDR2 comprising SEQ ID NO: 135; and LCDR1comprising SEQ ID NO:
 134. 29. The combination of claim 1, comprising anantibody molecule that binds IL1RAP, wherein said antibody molecule isselected from an antibody molecule comprising or consisting of avariable heavy chain domain (VH) and a variable light chain domain (VL)selected from the following: a VH domain comprising or consisting of theamino acid sequence of SEQ ID NO: 121 or an amino acid sequence havingat least 80%, 90%, 95%, 98% 99% identity thereto, and a VL domaincomprising or consisting of the amino acid sequence of SEQ ID NO: 122 oran amino acid sequence having at least 80%, 90%, 95%, 98% 99% identitythereto; and (ii) a VH domain comprising or consisting of the amino acidsequence of SEQ ID NO: 123 or an amino acid sequence having at least80%, 90%, 95%, 98% 99% identity thereto, and a VL domain comprising orconsisting of the amino acid sequence of SEQ ID NO: 124 or an amino acidsequence having at least 80%, 90%, 95%, 98% 99% identity thereto. 30.The combination of claim 1 comprising an antibody molecule that bindsCD47, wherein said antibody molecule inhibits the interaction betweenCD47 and SIRPα.
 31. The combination of claim 1 comprising an antibodymolecule that binds CD47, wherein said antibody molecule increasesphagocytosis of tumor cells.
 32. The combination of claim 1 comprisingan antibody molecule that binds CD47, wherein said antibody molecule isselected from: Hu5F9-G4; CC-90002; and ALX148.
 33. (canceled)
 34. Thecombination of claim 1, wherein the antibody molecules of thecombination are combined in a multispecific antibody.
 35. Thecombination of claim 34, wherein the combination comprises a firstantibody molecule that binds to CD70 and a second antibody molecule thatbinds to a leukemic stem cell target combined in a bispecific antibody.36. The combination of claim 1, wherein the antibody molecules of thecombination are co-formulated.
 37. (canceled)
 38. The combination ofclaim 1, wherein the antibody molecules of the combination are providedseparately. 39.-43. (canceled)
 44. The combination of claim 1, whereinthe combination additionally comprises an agent that inhibits SIRPαsignaling. 45.-49. (canceled)
 50. A combination comprising an antibodymolecule that binds to CD70 and an agent that inhibits SIRPα signaling.51. The combination of claim 50, wherein the agent that inhibits SIRPαsignaling is selected from: (i) an antibody molecule that binds CD47 andinhibits interaction between CD47 and SIRPα, (ii) antibody molecule thatbinds SIRPα and inhibits interaction between CD47 and SIRPα, (iii) anSIRPα-antibody molecule fusion protein. 52.-62. (canceled)
 63. A methodfor treating a malignancy in a human subject, said method comprisingadministering to the subject an effective amount of a combination asdefined in claim
 1. 64.-84. (canceled)